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VolumeVolume 3838 NumberNumber 11 MarchMarch 20172017 -associated diseases Now Available

Xpert® C. difficile BT Detection of toxigenic C. difficilewith binary toxin call-out in 47 minutes • The existing Xpert® C. difficile test detects binary toxin (i.e., cdt), but does not call-out the result independently of the tcdC deletion target used for presumptive 027 strain identification • The new Xpert C. difficile BT features a simple software change to call-out binary toxin detection independently of tcdC deletion^ • Binary toxin may be important because of the following: – Links to both disease severity and outcome1,2 – Strains, such as 033, are only positive for binary toxin and have been reported to cause C. difficile (CDI)3,4

* CE-IVD. Not for distribution in the U.S. ^ The new version of the test does not change the product itself (same probes, primers, and thermal cycling conditions) and the performance characteristics will be identical to the existing Xpert C. difficile test.

1 Bacci, et al. Emerg Infect Dis. Jun. 2011; 2 Stewart, et al. J Gastrointest Surg. 2013;(17):118-252; 3 Eckert, et al. New Microbes New Infect. 2014:(8);3:12-7; 4 Androga, et al. J Clin Microbiol. 2015;53:973-5

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904 Caribbean Drive PHONE (AUSTRALIA) 1800.107.884 Sunnyvale, CA 94089 USA PHONE (NEW ZEALAND) 0800.001.028 EMAIL [email protected] TOLL FREE +1.888.336.2743 PHONE +1.408.541.4191 FAX +1.408.541.4192 The Australian Society for Microbiology Inc. OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC. 9/397 Smith Street Fitzroy, Vic. 3065 Tel: 1300 656 423 Volume 38 Number 1 March 2017 Fax: 03 9329 1777 Email: [email protected] www.theasm.org.au Contents ABN 24 065 463 274 Vertical Transmission 2 For Microbiology Australia correspondence, see address below. Roy Robins-Browne 2 Editorial team Guest Editorial 3 Prof. Ian Macreadie, Mrs Jo Macreadie and Mrs Hayley Macreadie Bat-associated diseases 3 Glenn A Marsh Editorial. Board Dr Ipek Kurtböke (Chair) Prof. Wieland Meyer Focus 4 Prof. Mary Barton Prof. William Rawlinson In Prof. Linda Blackall Prof. Roy Robins-Browne : bat-borne paramyxoviruses 4 Dr Chris Burke Dr Paul Selleck Sarah Edwards and Glenn A Marsh Dr Narelle Fegan Dr David Smith Dr Gary Lum Ms Helen Smith Persistent or long-term infection in Australian 8 Dr John Merlino Dr Jack Wang Craig Smith Subscription rates Filoviruses and bats 12 Current subscription rates are available Amy J Schuh, Brian R Amman and Jonathan S Towner from the ASM Melbourne offi ce. Editorial correspondence The impact of novel discovery 17 Prof. Ian Macreadie/Mrs Jo Macreadie Ashley C Banyard and Anthony R Fooks Tel: 0402 564 308 (Ian) Email: [email protected] Under the Microscope 22 Published four times a year Menangle : one of the first of the novel from bats 22 in print and open access online by Peter D Kirkland Virus discovery in bats 25 Rebecca I Johnson and Ina L Smith Bats, and their role in health and disease 28 Unipark, Building 1, Level 1 Kristin Mühldorfer 195 Wellington Road, Clayton, Vic. 3168 http://microbiology.publish.csiro.au The interplay between viruses and the immune system of bats 30 Publishing enquiries Stacey Leech and Michelle L Baker Jenny Foster Bat and virus ecology in a dynamic world 33 Email: [email protected] David A Wilkinson and David TS Hayman Production enquiries Helen Pavlatos ASM Affairs 36 Email: [email protected] Bi-State Conference 2016: event report 36 Advertising enquiries Doug Walters Culture Media Special Interest Group (SIG) 38 Tel: 03 9545 8505 Mobile: 0419 357 779 FT-035 Food Microbiology, Standards Australia Committee 39 Email: [email protected] © 2017 The Australian Society for Microbiology Inc. Mycobacterium Special Interest Group (MSIG) 39 The ASM, through CSIRO Publishing, reserve all rights to the content, artwork and photographs in Microbiology Food Microbiology Special Interest Group (SIG) 40 Australia. Permission to reproduce text, photos and artwork must be sought from CSIRO Publishing. Vale Andrew Butcher 41 The Australian Copyright Act 1968 and subsequent Alison Vickery and the typing of staphylococci in Australia 42 amendments permit downloading and use of an article by an individual or educational institution for non- Vale Jennifer Taplin BSc (21/4/1929–21/10/2016) 43 commercial personal use or study. Multiple reproduction of any Microbiology Australia article in a study block is Vale Sue Dixon 44 governed by rights agreement managed by Copyright Agency Limited and fees may apply. Vale Joan Faoagali 45 Authors published in Microbiology Australia have the moral right under Australian law to be acknowledged as ASM Science Meets Business report 46 the creator. ISSN 1324-4272 ASM @ Science Alive! 2016 47 eISSN 2201-9189 While reasonable effort has been made to ensure the accuracy of the content, the Australian Society for Microbiology, CSIRO, and CSIRO Publishing accept no responsibility for any loss or damage from the direct or indirect use of or reliance on the content. The opinions expressed in articles, letters, and advertisements in Microbiology Australia are not necessarily those of the Australian Society for Microbiology, the Editorial Board, CSIRO, and CSIRO Publishing. Cover image: Photograph kindly provided by Prof. Wanda Markotter and Stewart McCulloch, Centre for Viral Zoonoses, University of Pretoria, RSA showing Miniopterus natalensis, taken in Meletse, Limpopo Province, RSA.

MICROBIOLOGY AUSTRALIA • MARCH 2017 1 Vertical Transmission

the Sands Expo & Convention Centre in Singapore from 17–21 July. Over the span of 5 days, this meeting will bring together three congresses: the 15th International Congress of Bacteriology and Applied Microbiology, the 15th International Congress of Mycology and Eukaryotic Microbiology, and the 17th International Congress of (http://www.iums2017singapore.com/). The scope of this meeting guarantees there will be something for Roy Robins-Browne everyone. President of ASM September will be a particularly busy month for members, with the back-to-back 2017 Tri-State Scientific Meeting and Parasitology As this is my first communication for 2017, I will begin by wishing Masterclass in Darwin, NT, from 22–25 September (http:// you a happy and prosperous New Year. theasm.org.au/assets/ASM-Society/Current-Event-Flyers/Tri-State- ASM members have much to look forward to in 2017 as far as 2017-Save-the-Date-flyer.pdf), followed by BacPath14: The Molec- scientific meetings are concerned. ular Biology of Bacterial , which will take place from 25–28 September at the Adelaide Hills Convention Centre in From 13–15 February, the inaugural Australian Microbial Ecology Hahndorf, South Australia (http://bacpath.org/). (AusME) meeting was held at the Peter Doherty Institute for Infection and Immunity in Melbourne. Please check http:// Please make every effort to attend at least one of these meetings. If ausme-microbes.org.au/ for details. you haven’t been to a scientific conference for some time, you may fi fi This year's Annual Scientific Meeting will take place at the Hotel be surprised to nd how rewarding and ful lling they are from both Grand Chancellor in Hobart from 2–5 July (http://www.asmmeet a personal and professional point of view. ing.theasm.org.au/). The National Scientific Advisory Committee ASM Council is also working on various reforms to our bylaws to and Conference Local Organising Committee have prepared a make it easier for individuals to become Professional Members varied and stimulating scientific program, together with some (MASM) and Fellows (FASM) of ASM, and for all members to exciting social events based on Tasmania’s renowned foods and contribute to the ongoing development of our Society. I will have beverages. more to say on this in my next communication. In the meantime, For the more adventurous, the International Union of Microbio- please explore the links above and select the conference(s) you will logical Societies (IUMS), of which ASM is a member, will meet at attend this year.

2 10.1071/MA17001 MICROBIOLOGY AUSTRALIA * MARCH 2017 Guest Editorial

Bat-associated diseases

decade has resulted in the detection and isolation of many new viruses from bats, some of which have been associated with dis- eases of and . In Australia, apart from , Glenn A Marsh Australian Bat Lyssavirus and Menangle virus are two examples of Australian Health Laboratory bat-borne viruses that have resulted in disease. In addition to these CSIRO Health and Biosecurity two viruses, many other viruses, particularly those of the Para- 5 Portarlington Road East Geelong, Vic. 3219, Australia myxoviridae family have been isolated from Australian fruit bats, Tel: +613 5227 5125 Fax: +613 5227 5555 including Cedar virus, a seemingly non-pathogenic member of the Email: [email protected] .

Other virus families that have been found in bats include Rhabdo- viridae, , , , Togavir- Emerging infectious diseases pose asignificant threat to humanand idae, , Bunyaviridae, , , animal health. Increasingly, emerging and re-emerging infectious , , Retroviridae and . diseases are of zoonotic origin and are derived from wildlife. Bats have been identified as an important reservoir of zoonotic viruses The articles in this issue highlight some of more serious of these belonging to a range of different virus families including SARS- viruses that have spilled over into humans. The recent outbreak of Coronavirus, virus, Hendra virus, , virus in west demonstrates the potential of these bat and Ebola virus. viruses to result in significant disease. One hypothesis of the origin of this outbreak was that a two-year-old boy who died in Bats have been reported to harbour more zoonotic viruses per December 2013 in the village of Meliandou, Guéckédou Prefecture, than and are now recognised as a significant source Guinea, was the index case. It has been suggested that the boy of zoonotic agents. The spill-over of virus from bats to humans, became infected whilst playing around a tree that was the home of often through an intermediate, results in rare however, a large colony of Angolan free-tailed bats. The presence of virus in the consequences of infection are often fatal disease. this bat colony was not able to be confirmed as the tree had been Bats are belonging to the order Chiroptera, and are the burnt and the bats moved on prior to any investigation. The single only that has true flight capabilities. Over 1200 species of introduction of virus into humans resulted in an outbreak that bats exist worldwide, being found in all continents except for lasted over two years and resulted in over 28 000 cases and 11 000 Antarctica. This species diversity is second only to rodents. Bats deaths. have unique and specialised capabilities including echolocation, A greater understanding the infectious diseases present in bats hibernatioflight. and the potential of these to cause disease in humans and animals For many reasons bats are the perfect reservoir species for infec- is important to mitigate the risks. Additional work globally is tious diseases. They often live in large colonies or roosts, are able to needed to understand how to identify and characterise these travel considerable distances and they enjoy remarkable longevity novel infectious agents and potentially proactively develop vac- for their body size. Human activities, such as , are cines and therapeutics that could be deployed if human disease was increasing interactions between bats, humans, and livestock, there- to eventuate. by increasing the opportunities for zoonotic spill-over. For these reasons, bats present a significant potential source of emerging Biography infectious diseases. Glenn Marsh is a Senior Research Scientist and Team Leader for The discovery of Hendra virus in fruit bats in Australia and spill- Dangerous Pathogens in CSIRO. His research interests include overs resulting in deaths of both horses and humans began a global development of animal models for high consequence viruses and renaissance in virus discovery from bats. Research over the past using molecular tools to identify virulence determinants.

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17002 3 In Focus

Henipaviruses: bat-borne paramyxoviruses

Glenn A Marsh Sarah Edwards Australian Animal Health Laboratory Australian Animal Health Laboratory CSIRO Health and Biosecurity CSIRO Health and Biosecurity 5 Portarlington Road 5 Portarlington Road East Geelong, Vic. 3219, Australia East Geelong, Vic. 3219, Australia Tel: +61 3 5227 5125 Tel: +61 3 5227 5125 Fax: +61 3 5227 5555 Fax: +61 3 5227 5555 Email: [email protected]

Found on every continent except Antarctica, bats are one Following initial HeV outbreaks, the closely related NiV emerged of the most abundant, diverse and geographically wide- in 1998 amongst the and human population in Peninsular spread globally, making up approximately . During the outbreak, 105 people died from 265 cases of 20% of all known extant mammal species1,2. Noted for being infection with febrile illness and encephalitis15. The disease was the only mammal with the ability of powered flight, bats also later recorded in Singapore, transmitted via the importation of constitute the order Chiroptera (from the Ancient Greek infected swine from the outbreak region of Malaysia. Over 1 million meaning ‘hand wing’), which is further divided into two were culled in a large-scale effort to eradicate the causative suborders: Megachiroptera known as or flying – an effort that has proven successful in this region11,16. foxes, and Microchiroptera comprising of echolocating Three years later, disease with hallmarks of febrile neurological microbats1,3. symptoms resulted in the death of 9 people in a Bangladeshi village. An investigation into subsequent outbreaks showed a Known for their important role in the of flora, seed reaction of to NiV antigens17, however, unlike the dispersal and insect population control, bats also play a key role in the spread and perseverance of many notable zoonotic viruses Table 1. Occurrence of Hendra virus disease events recorded in which cause severe disease and potentially fatal outcomes for Australia, 1994–2016. humans, livestock and many other species1,4. In particular, mega- Year Number of Confirmed Confirmed Fatality HeV cases in cases in rate in bats belonging to the genus , are important natural reser- events horses humans humans % voirs for many significant pathogenic viruses such as Hendra virus 1994 2 22 3 66 (HeV), Nipah virus (NiV), SARS Coronavirus, Australian bat Lyssa- 1999 1 1 – – virus and Menangle virus4–9. 2004 2 2 1 0 2006 2 2 – – The emergence of Henipaviruses 2007 2 2 – – HeV was first observed in 1994 during a severe respiratory disease 2008 2 8 2 50 outbreak in , Australia, in which 13 of 21 infected horses 2009 2 6 1 50 died within a two week period10. Additionally, two humans who 2010 1 1 – – frequently had extensive contact with the affected horses were confirmed as infected, with one later dying as a result10,11. Since the 2011 18 23 – – emergence of HeV, sporadic outbreaks have occurred within the 2012 8 10 – – equine population of Australia, which have resulted in four human 2013 7 7 – – fatalities, as well as the death or euthanasia of over 84 horses and 2014 1 1 – – two dogs12,13 (Table 1). In late 2012 an HeV , based on the 2015 1 1 – – G glycoprotein, was released for use in horses14. Since this time, 2016 1 1 – – all identified cases of HeV have been in unvaccinated horses.

4 10.1071/MA17003 MICROBIOLOGY AUSTRALIA * MARCH 2017 In Focus

Malaysian NiV outbreak, pigs were not the intermediate and am- from a bat in Ghana (Kumasi virus)33 and the other from a Chinese plifying . Instead, human-to-human and bat-to-human trans- rat (Mòjiang virus)34. mission routes were involved in NiV outbreaks occurring within There is evidence that less pathogenic Henipaviruses may also be the region – events not previously observed of the circulating in Australia, exemplified by the newly identified CedPV henipaviruses11. Furthermore, the more recent Nipah outbreaks virus35. This was observed to share a similar size and demonstrated a notably higher fatality ratio of 74%, compared to organisation with HeV and NiV, showed cross-reactivity with heni- 38.5% for the Malaysia outbreak15,18. pavirus antigens, and also used the same host cell receptor for During 2014, severe illness among humans and horses in southern infection. Despite these similarities, disease was not observed with 19 Philippines was attributed to henipavirus infections . Seventeen CedPV infection in various animal models in which a lethal disease fi cases met the case de nition with 2 survivors, of these seven had results from HeV or NiV challenge. This is thought to be due to participated in horse slaughtering and horse meat consumption. differences in its phosphoprotein gene in which it lacks RNA editing Horse-to-human and human-to-human transmission occurred. sequences and a highly conserved V open reading frame35, whose Additionally, although recovery from HeV and NiV infection is product is responsible for modulating the host innate 32 possible, relapsed has been shown to occur in 3–7% immune response . of individuals anywhere from months to years following recovery from acute infection20. The molecular biology of Henipaviruses HeV, NiV and CedPV belong to the genus Henipavirus (order Bats as hosts to the Henipaviruses , family Paramyxoviridae). HeV and NiV were the first observed zoonotic Paramyxoviruses resulting from spill- Following the 1994 Brisbane HeV outbreak, a large serological over events of Pteropid bats36. High virulence, human susceptibil- survey of horses showed no evidence of HeV infection outside the ity, broad host species range, and a lack of human and index property21, suggesting horses were not commonly infected therapeutic treatment has resulted in HeV and NiV being restricted with these viruses. An extensive sampling exercise of native and to four (BSL4) containment – the only Paramyxo- introduced animal species was carried out with antibodies to HeV viruses to be restricted to this level35,37,38. The lack of a human being detected in all four mainland Australian flying fox species: vaccine and licensed therapeutics necessitates on-going research the Black flying fox (Pteropus alecto), Grey-headed flying fox to understand these viruses and the virulence determinants. (P. poliocephalus), Spectacled flying fox (P. conspicilla- tus) and the Little red flying fox (P. scapulatus)9,22. Further research has Members of the Paramyxoviridae family are enveloped, non- resulted in isolation of HeV directly from pteropid bats23,24. segmented negative-stranded RNA viruses which can cause a range of respiratory and systemic disease in both humans and animals. Following the 1998 Malaysian NiV outbreak, investigations of bat Paramyxovirus share similar ultrastructural appearance, colonies across a large area of Peninsular Malaysia revealed evi- and organisation of genes contained within a genome which can dence of NiV antibodies in the two pteropid species found in range from 15–20 kilobases (kb), with henipaviruses having a Malaysia: the Island flying fox (P. hypomelanus) and the Malayan genome size of 18.2 kb39,40. Henipavirus genomes encode six flying fox (P. vampyrus)25,26. NiV was also isolated from urine and major . Reading in order from 3’ to 5’ on the antigenome partially eaten fruit collected from a colony of P. hypomelanus on they are: nucleocapsid protein (N), phosphoprotein (P), matrix Tioman Island, off the coast of Peninsular Malaysia27. protein (M), fusion protein (F), attachment glycoprotein (G), and Following the emergence of NiV in and Bangladesh in 2001, RNA-dependent RNA polymerase (L)39,41 (Figure 1). P. giganteus,aflying fox found across the , was Essential for the replication and of paramyxoviruses, shown to have antibodies to NiV17. To date, no NiV isolate has been the genomic RNA is encapsidated by the N protein, the most reported from bats in either Bangladesh or India. abundant protein in the purified virion. The N protein associates Although disease associated with HeV and NiV has only been with both the P and L proteins, forming the ribonucleoprotein observed in Australia and South and South-east Asia, serological (RNP) complex, and it is this complex that serves as a template and molecular evidence of Henipavirus have been reported from for replication42,43. Following transcription and of many different countries, including Africa28–31 and Asia32. This viral proteins, the M protein mediates the association of the plasma includes 2 full genome sequences that have been obtained, one membrane with the ribonucleoprotein and glycoproteins F and

MICROBIOLOGY AUSTRALIA * MARCH 2017 5 In Focus

(a) ‘helper’ which each encode a gene required for viral Orange: N replication and gene expression. The genes encoded on these Green: Phophoprotein P expression vectors are present as cDNA, and are therefore not Yellow: Matrix protein M 50,52,53 Purple: Fusion protein F infectious . Blue: Attachment glycoprotein G Red: RNA polymerase L Grey: Viral genomic RNA Future perspectives The infection of HeV and NiV in horses and pigs, respectively, demonstrate the potential threat of dangerous bat-borne zoonotic (b) agents to livestock as the intermediate and amplifying host for NPM FG L transmission to humans. Factors contributing to the emergence of Figure 1. A simplified schematic of the henipavirus virion (a) and these viruses into the human population include deforestation, organisation of the henipavirus genome (b). closer living proximities between humans and bats, and a greater yield demand and turnover of livestock farming practices. Consid- G. After assembly, the newly matured virion is then ready to bud and ering the close human interaction with livestock within current 44 be released from the host cell . farming practices globally, livestock interaction with the bat pop- ulation is of high importance for both intervention and counter- The attachment glycoprotein (G glycoprotein/HN haemaglutinin- measure strategies to prevent incidences of human infection. /H haemaglutinin; depending on the virus), is es- In addition to surveillance of both bat populations and any circu- sential for the initiation of infection by recognising and attaching to lating zoonotic agents they may harbour, further study of these membrane-bound host cell receptors. In the case of the henipa- viruses on a molecular biology and immunology scale is needed. viruses, the G glycoprotein attaches to receptors ephrin B2 and – Broadening our understanding of key factors such as bat immunity, B345 47. The binding of the G protein to the ephrin host cell virus transmission, host response and pathogenesis, could receptor facilitates the F protein-dependent fusion event, however make a significant contribution to the development of vaccines the exact signalling cascade from the bound G glycoprotein to the and therapeutic treatments for these highly pathogenic bat-borne fusion-competent F protein is not well understood47. Due to infections. the enveloped nature of the paramyxoviruses, it is essential that fusion-competent F proteins generate membrane fusion between the virus envelope and the plasma membrane of the receptive References host cell through a pathway of conformational changes in the viral 1. Calisher, C.H. et al. (2006) Bats: important reservoir hosts of emerging viruses. Clin. Microbiol. Rev. 19 – envelope proteins38. ,531 545. doi:10.1128/CMR.00017-06 2. Teeling, E.C. et al. 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(1996) Negative findings from serological studies of equine the envelope glycoproteins of Hendra virus. Virology 290,121–135. doi:10.1006/ morbillivirus in the horse population. Aust. Vet. J. 74,241–243. viro.2001.1158 doi:10.1111/j.1751-0813.1996.tb15412.x 46. Bonaparte, M.I. et al. (2005) Ephrin-B2 ligand is a functional receptor for Hendra 22. Young, P.L. et al. (1996) Serologic evidence for the presence in Pteropus bats of virus and Nipah virus. Proc. Natl. Acad. Sci. USA 102, 10652–10657. doi:10.1073/ a paramyxovirus related to equine morbillivirus. Emerg. Infect. Dis. 2,239–240. pnas.0504887102 doi:10.3201/eid0203.960315 47. Aguilar, H.C. and Iorio, R.M. (2012) Henipavirus membrane fusion and . et al 23. Halpin, K. . (2000) Isolation of Hendra virus from pteropid bats: a natural Curr. Top. Microbiol. Immunol. 359,79–94. doi:10.1007/82_2012_200 reservoir of Hendra virus. J. Gen. Virol. 81, 1927–1932. doi:10.1099/0022- 48. Skiadopoulos, M.H. et al. (2003) The genome length of human parainfluenza 1317-81-8-1927 virus type 2 follows the rule of six, and recombinant viruses recovered from non- et al PLoS One 24. Smith, I. . (2011) Identifying Hendra virus diversity in pteropid bats. polyhexameric-length antigenomic cDNAs contain a biased distribution of cor- 6 , e25275. doi:10.1371/journal.pone.0025275 recting . J. Virol. 77,270–279. doi:10.1128/JVI.77.1.270-279.2003 25. Shirai, J. et al. (2007) Nipah Virus Survey of Flying Foxes in Malaysia. Jpn. Agric. 49. Vulliémoz, D. and Roux, L. (2001) ‘Rule of six’: how does the Sendai virus RNA Res. Q. 41,69–78. doi:10.6090/jarq.41.69 polymerase keep count? J. Virol. 75, 4506–4518. doi:10.1128/JVI.75.10.4506- 26. Yob, J.M. et al. (2001) in bats (order Chiroptera) in 4518.2001 peninsular Malaysia. Emerg. Infect. Dis. 7, 439–441. doi:10.3201/eid0703.017312 50. Yoneda, M. et al. (2006) Establishment of a Nipah virus rescue system. Proc. Natl. Acad. Sci. USA 103, 16508–16513. doi:10.1073/pnas.0606972103 27. Chua, K.B. et al. (2002) Isolation of Nipah virus from Malaysian Island flying- foxes. Microbes Infect. 4,145–151. doi:10.1016/S1286-4579(01)01522-2 51. Kovacs, G.R. et al. (2003) Enhanced genetic rescue of negative-strand RNA viruses: use of an MVA-T7 RNA polymerase vector and DNA replication inhibitors. 28. Iehlé, C. et al. (2007) Henipavirus and Tioman virus antibodies in pteropodid J. Virol. Methods 111,29–36. doi:10.1016/S0166-0934(03)00132-0 bats, Madagascar. Emerg. Infect. Dis. 13, 159–161. doi:10.3201/eid1301.060791 52. Brown, D.D. et al. (2005) ‘Rescue’ of mini-genomic constructs and viruses by 29. Drexler, J.F. et al. (2009) Henipavirus RNA in African bats. PLoS One 4, e6367. combinations of morbillivirus N, P and L proteins. J. Gen. Virol. 86, 1077–1081. doi:10.1371/journal.pone.0006367 doi:10.1099/vir.0.80804-0 30. Hayman, D.T. et al. (2008) Evidence of henipavirus infection in West African fruit 53. Freiberg, A. et al. (2008) Establishment and characterization of -driven bats. PLoS One 3, e2739. doi:10.1371/journal.pone.0002739 minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed et al 31. Pernet, O. . (2014) Evidence for henipavirusspillover into human populations generation of functional paramyxoviral RNA. Virology 370,33–44. doi:10.1016/ Nat. Commun. 5 in Africa. , 5342. doi:10.1038/ncomms6342 j.virol.2007.08.008 32. Baker, K.S. et al. (2013) Novel, potentially zoonotic paramyxoviruses from the African straw-colored fruit bat helvum. J. Virol. 87,1348–1358. doi:10.1128/JVI.01202-12 Biographies 33. Drexler, J.F. et al. (2012) Bats host major mammalian paramyxoviruses. Nat. Sarah Edwards is a PhD student based at the CSIRO Australian Commun. 3, 796. doi:10.1038/ncomms1796 Animal Health Laboratory. Her project is investigating the mole- 34. Wu, Z. et al. (2014) Novel Henipa-like virus, Mojiang Paramyxovirus, in rats, China, 2012. Emerg. Infect. Dis. 20, 1064–1066. doi:10.3201/eid2006.131022 cular mechanisms of neurological invasion of the henipaviruses in 35. Marsh, G.A. et al. (2012) Cedar virus: a novel Henipavirus isolated from Australian a mouse model. bats. PLoS Pathog. 8, e1002836. doi:10.1371/journal.ppat.1002836 Glenn Marsh 36. Mortlock, M. et al. (2015) Novel Paramyxoviruses in Bats from Sub-Saharan is a Senior Research Scientist and Team Leader for Africa, 2007–2012. Emerg. Infect. Dis. J. 21, 1840. doi:10.3201/eid2110.140368 Dangerous Pathogens in CSIRO. His research interests include 37. Craft, W.W. Jr and Dutch, R.E. (2005) Sequence motif upstream of the Hendra development of animal models for high consequence viruses and virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing. Virology 341, 130–140. doi:10.1016/j.virol.2005.07.004 using molecular tools to identify virulence determinants.

MICROBIOLOGY AUSTRALIA * MARCH 2017 7 In Focus

Persistent or long-term coronavirus infection in Australian bats

in the Atlantic Forest Biome, Brazil8,14. However, in spite of these investigations and the potential serious consequences of these high-profile pathogens, knowledge of their ecology is still limited. For example, it is still unknown how these are maintained, amplified or controlled by their chiropteran hosts15.

Previous studies by Drexler et al.15 identified two peaks of ampli- Craig Smith fication of coronaviruses, characterised by increased virus concen- University of Queensland Brisbane, Qld, Australia tration and increased detection rates, upon the formation of a Email: [email protected] colony of Myotis myotis in Germany and following parturition. It was hypothesised that the initial peak was probably due to the formation of a colony of sufficient size and density to allow the When the World Health Organization declared the end of establishment of a viral transmission cycle in susceptible bats. the global outbreak of severe acute respiratory syndrome The second peak, after parturition, was associated with the intro- (SARS) on the 5 July 2003, more than 8000 cases with over duction of susceptible bats, newborn pups who had lost their 800 fatalities had been reported in 32 countries worldwide perinatal protection but not yet mounted their own adaptive 15 fi and financial costs to the global economy were close to immunity . In another attempt to better de ne the epidemiology et al 16 $US40 billion1,2. Coronaviruses were identified as being of coronaviruses, Lau . marked 511 Chinese horseshoe bats Rhinolophus responsible for the outbreaks of both SARS and Middle ( spp) from 11 sites and recaptured 113 (22%). From East respiratory syndrome (MERS, the latter in 2013). Sub- this study it was estimated that viral clearance occurred between sequently, bats (order Chiroptera) were identified as the two and 16 weeks after infection and suggested that coronaviruses natural hosts for a large number of novel and genetically in Chinese horseshoe bats caused an acute self-limiting infection fi diverse coronaviruses, including the likely ancestors to associated with weight loss. It was also identi ed that the peak SARS-like and MERS-like coronaviruses3–8. activity for coronaviruses was during spring, soon after hibernation, and that mating and feeding activity may have facilitated the spread Coronaviruses, of the order , family Coronaviridae, are of the virus within and between roosts. the largest known non-segmented, single stranded, positive sense RNA viruses (28 to 32 kb). They have large projections protruding Persistent or long-term infection of Australian from the envelope that are formed by trimers of the and when viewed by electron microscopy form the characteristic bat coronavirus ‘crown’ that gave rise to the family’s name. Coronaviruses can Subsequent to these ecological studies, we identified four putative cause a range of syndromes including respiratory and gastroenteric novel coronaviruses (two Alpha- and two )in disease in humans and respiratory, gastroenteric, neurological and seven species of Australian bats17,18. One of these species (Myotis hepatic disease in animals, often with significant economic con- macropus, Figure 1), had individuals infected with a putative novel sequences. Respiratory and faecal-oral transmission are common Alphacoronavirus (detection of coronavirus RNA in faeces from but biological vectors are not known. Pigs, cats and domestic fowl bats enrolled in a mark-recapture study) over periods of up to may become persistently infected and shed virus from the enteric 11 weeks, supporting the hypothesis for persistent or long-term tract, many doing so for a lifetime9–13. infection as a method of maintaining coronaviruses in bats17,19.

The ecology of bat coronaviruses around This period of infection (up to 11 weeks) was consistent with that observed by Lau et al.16 of between two and 16 weeks. the world However, whereas Lau et al.16 suggested that SARS-Rh-BatCoV Surveillance and identification of bat coronaviruses continues caused an acute, self-limiting infection in individual Chinese horse- to occur around the world, most recently with the detection of shoe bats, our interpretation would be that the Australian Alpha- SARS-like and MERS-like coronaviruses in bats in Korea, and the coronavirus appears capable of a persistent or long-term infection demonstration of genetically diverse clusters of bat coronaviruses of bats. Persistent infection has previously been suggested as

8 10.1071/MA17004 MICROBIOLOGY AUSTRALIA * MARCH 2017 In Focus

playing a role in the maintenance of coronaviruses in populations of real and there are true variations in patterns of infection for bats, as it does for other coronaviruses, including feline corona- different species of coronaviruses and bats, or it could be that viruses (FECV) where it has been shown that naturally infected cats the limited rate of recapture of infected bats in the study by Lau shed FECV intermittently for periods up to 10 months but et al.16 precluded an accurate interpretation of infection. Whilst some (~15%) become chronic shedders, doing so for years or a Lau et al.16 made a significant effort in marking 511 bats, only 113 lifetime12,13,20–22. (22%) bats were recaptured and coronavirus was only ever detected in 63 of the 511 bats (12%). Of these 63 bats, shedding of corona- The apparent discrepancy between an acute infection observed by virus was detected in only one bat on more than one occasion Lau et al.16 and a persistent infection interpreted from this study’s (two weeks apart) and 10 bats which were detected shedding results requires clarification. It is possible that the discrepancy is coronavirus at one sampling event were not detected shedding when recaptured (between 4 and 16 months later), providing an interpretation of an infectious period of between 2 and 16 weeks (4 months). Conversely, whilst only employing 52 marked bats, our study had a viral prevalence of 54% (28 bats) and a recapture rate of 81% (42 bats). The weekly sampling events and the affinity of bats for their roost, provided a unique opportunity to frequently recapture marked individuals that were shedding coronavirus (Figure 2). This increased probability of recapturing bats allowed interpretation of the pattern of infection for our longitudinal study and reasonably suggested a persistent infection of coronaviruses in Australian bats19.

Previous studies suggested that physiological stress associated with pregnancy and lactation was a risk factor for increased seroprev- Myotis macropus Figure 1. A female and her 2-week-oldpup. This female 26,27 had an implantable radio frequency identification transponder, more alence of virus infections in bats . Similarly, a correlation be- commonly known as a ‘microchip’, subcutaneously implanted on the tween the detection of coronaviruses in female bats associated with dorsum during Week 2 of the mark-recapture study, when she was 28,29 identified (by palpation of the abdomen) as being pregnant. She was maternity colonies has also been established . The colony used recaptured on Week 4 and was again identified as being pregnant, on in our study had been selected for its ease of access and the Week 5 shehad given birth andthe pup wasattached. On Week 7 the pup was still attached and they were both photographed. When recaptured high affinity of bats to the roost, providing a successful recapture on Week 12 the pup was no longer attached and was assumed to have rate. It was opportunistically and irregularly sampled over the weaned, roosting separately with the other weaned pups that were observed in the colony19. Photograph courtesy of Steve Parish. previous year, with a coronavirus RNA detection prevalence of

(a) (b)

Figure 2. A collapsible bat trap. The collapsible bat trap (a), commonly known as a harp trap was developed by Tidemann and Woodside23 based on the original designs of Constantine24 and Tuttle25. The trap is a common tool used for the capture of insectivorous bats and is best placed in the natural flight path of bats, including; roads, trails, streams and roost entrances. The trap is light and portable and can be set up in 5 minutes by a single person. The author removing captured bats from the bag of a harp trap (b).

MICROBIOLOGY AUSTRALIA * MARCH 2017 9 In Focus

between 30% (19–45%, 95%CI) one year prior to the commence- up to 11 weeks, supports the hypothesis of persistent infection ment of the mark-recapture study, and 0% (0–15%, 95%CI) of coronaviruses in some individual bats. Patterns of infection in three months prior. Only during the first sampling event did we other individuals are suggestive of intermittent identify that the majority of female adults (88%) were pregnant and (of persistently infected bats) but could also be interpreted as an that the study site should be considered a maternity colony. acute infection (lack of detection in this species precluded In agreement with other studies15,28,29, it appears that the site had distinguishing between the two). While taking care to avoid over- an increased prevalence of coronavirus when used as a maternity interpretation, a persistently infected bat could become a chronic colony (during the mark-recapture study and exactly one year shedder (as it does for other coronaviruses, including feline cor- prior), as opposed to other times (three months prior) when no onaviruses). This chronic shedder could potentially be the source coronavirus was detected and no pregnant females were observed. of infection to a maternal colony upon its formation.12,13,20–22. However, modelling the presence or absence of coronavirus (using logistic regression) did not show any association with the animal Conclusion risk factors pregnancy or lactation status, and suggests that phys- iological or environmental stressors are not driving coronavirus When discussing the infection dynamics of bat coronaviruses it infection in Australian bats19. would be remiss to ignore the unique biology of bats, the only mammals with the ability for true sustained flight. Flight has Alternatively, migration of bats has been shown to play a role in the previously been linked with viral infection dynamics of bats. It has maintenance of viruses; immigration allows the maintenance of an been suggested that elevated metabolism and body temperature 30,31 infection through newly introduced susceptible individuals . generated during daily cycles of flight was analogous to a febrile However, the population of bats used in our study appeared response in other mammals and on an evolutionary scale produced relatively closed with the population size remaining between 72 a diversity of viruses more tolerant of the fever response32. Also, fi and 101 bats and apparent high delity to the roost site (assumed that reactive oxygen species (a by-product of metabolism) placed from the high recapture rate of marked bats, 81%). It is therefore positive selective pressure on a high proportion of the genes in unlikely that immigration of susceptible bats was responsible for the DNA damage checkpoint. These flight induced adaptions Alphacoronavirus the maintenance of the in this relatively small may have had inadvertent effects on bat immune function and and closed population. Throughout a three year study, Drexler expectancy33. et al.15 observed that strong and specific amplification of RNA viruses, including coronaviruses, occurred upon colony formation By themselves these adaptations in response to the evolution of fl and following parturition. They suggested that the initial peak, ight could have an effect on viral infection dynamics, but the fl upon colony formation, was due to the massing of enough sus- product of ight itself, (general frequent and long distance move- 34,35 ceptible bats to establish a viral transmission cycle and that the ment ) would not only have the effect of spreading the virus second amplification peak was associated with the introduction of a into new populations, but would surely have some selective pres- susceptible subpopulation of newborn pups losing their perinatal sure on viruses hosted by bats. Whilst it is reasonable to assume immunity. Interestingly, in our Australian study, we also observed that increased viral prevalence is the result of the congregation of two apparent peaks of infection during a three-month study of susceptible bats (in a maternal colony or otherwise), conversely, a maternal colony. Whilst bats occupied this colony irregularly a survival strategy is required for the coronaviruses during periods ’ fl throughout the year, it was upon the formation of the maternity of its host s dispersal (when ight has afforded the bats the ability colony that the first peak was observed, coinciding with the to spread out over large areas). Could it also be that whilst bats 15 fl observations of Drexler et al. . The second peak followed two have adapted to the evolution of ight by controlling the damage months later but cannot be conclusively attributed to maternal of DNA and effects of viral infection, viruses have co-evolved to antibody loss in the subpopulation of newborn pups as none were survive periods of time when susceptible hosts are sparse? Is this sampled. It is probable that our inability to sample newborn pups the difference that fundamentally drives different transmission (in an endeavour to reduce stress on them and their mothers) dynamics of coronaviruses in bat populations and requires a reduced our ability to identify this risk factors responsible for persistent infection for bat coronaviruses to endure? Jeong et al 36 the second peak (loss of perinatal immunity in newborn pups). . recently attempted to answer this question by developing Myotis macropus Also, whilst our study had a very successful recapture rate (82%), epidemic models using the mark-recapture 17,19 the overall sample size (52) was too small and likely precluded data , and found that both persistently and transiently infected us from identifying significant associations for the detection of bats were required for maintenance of coronaviruses. coronaviruses. Together, these studies support the hypothesis for the existence Our identification that individual Australian Myotis macropus were of persistently infected bats and demonstrate an important role infected with a novel putative Alphacoronavirus over periods of that these individuals play in the maintenance of coronaviruses.

10 MICROBIOLOGY AUSTRALIA * MARCH 2017 In Focus

A better understanding of a viral transmission cycle is an important 17. Smith, C.S. et al. (2016) Coronavirus infection and diversity in bats in the EcoHealth 13 – step towards breaking it and armed with this knowledge we may Australasian region. ,72 82. doi:10.1007/s10393-016-1116-x 18. Smith, C.S. et al. (2010) Sampling small quantities of blood from microbats. Acta be better prepared to prevent the next global pandemic of a bat Chiropt. 12,255–258. doi:10.3161/150811010X504752 coronavirus. 19. Smith, C.S. (2014) Australian bat coronaviruses. Brisbane: University of Queensland. Acknowledgements 20. Chu, D.K. et al. (2006) Coronaviruses in bent-winged bats (Miniopterus spp.). J. Gen. Virol. 87, 2461–2466. doi:10.1099/vir.0.82203-0 The author gratefully acknowledges; contributions to the project 21. Tang, X.C. et al. (2006) Prevalence and genetic diversity of coronaviruses in bats by Carol de Jong, Joerg Henning, Joanne Meers, Linfa Wang and from China. J. Virol. 80,7481–7490. doi:10.1128/JVI.00697-06 Hume Field, funding from the Australian Biosecurity Co-operative 22. Addie, D.D. et al. (1995) Risk of feline infectious peritonitis in cats naturally Am. J. Vet. Res. 56 – Research Centres and the Wildlife Exotic Disease Preparedness infected with feline coronavirus. , 429 434. 23. Tidemann, C.R. and Woodside, D.P. (1978) A collapsible bat-trap and a compar- Program, and the in-kind contribution of the Biosecurity Sciences ison of results obtained with the trap and with mist-nets. Aust. Wildl. Res. 5, Laboratory, Biosecurity Queensland, Department of Agriculture 355–362. doi:10.1071/WR9780355 and Fisheries. 24. Constantine, D.G. (1958) An automatic bat-collecting device. J. Wildl. Manage. 22,17–22. doi:10.2307/3797291 25. Tuttle, M.D. (1974) An improved trap for bats. J. Mammal. 55,475–477. References doi:10.2307/1379025 1. Centers for Disease Control and Prevention. (2003) Update: outbreak of severe 26. Plowright, R.K. et al. (2008) Reproduction and nutritional stress are risk factors — MMWR Morb. Mortal. Wkly Rep. acute respiratory syndrome worldwide, 2003. for Hendra virus infection in little red flying foxes (Pteropus scapulatus). Proc. 52 – ,241 246. Biol. Sci. 275,861–869. doi:10.1098/rspb.2007.1260 2. Lee, J.W. and McKibbin, W.J. (2004) Estimating the global economic cost of 27. Breed, A.C. et al. (2011) Evidence of endemic Hendra virus infection in flying- Learning from SARS: preparing for the next disease outbreak: workshop SARS. In foxes (Pteropus conspicillatus) – implications for disease risk management. summary et al (Knobler, S. . eds). Washington, DC: National Academies Press. PLoS One 6, e28816. doi:10.1371/journal.pone.0028816 3. Drosten, C. et al. (2003) Identification of a novel coronavirus in patients with 28. Gloza-Rausch, F. et al. (2008) Detection and prevalence patterns of group I severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967–1976. doi:10.1056/ coronaviruses in bats, Northern Germany. Emerg. Infect. Dis. 14,626–631. NEJMoa030747 doi:10.3201/eid1404.071439 4. Zaki, A.M. et al. (2012) Isolation of a novel coronavirus from a man with 29. Pfefferle, S. et al. (2009) Distant relatives of severe acute respiratory syndrome in Saudi Arabia. N. Engl. J. Med. 367, 1814–1820. doi:10.1056/ coronavirus and close relatives of in bats, Ghana. Emerg. NEJMoa1211721 Infect. Dis. 15, 1377–1384. doi:10.3201/eid1509.090224 5. Li, W. et al. (2005) Bats are natural reservoirs of SARS-like coronaviruses. Science 30. Plowright, R. et al. (2011) Urban habituation, connectivity, and stress synchrony: 310,676–679. doi:10.1126/science.1118391 Hendra virus emergence from flying foxes (Pteropus spp.). EcoHealth 7, 6. Lau, S.K. et al. (2005) Severe acute respiratory syndrome coronavirus-like S36–S37. virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. USA 102, 14040–14045. 31. Drexler, J.F. et al. (2011) Amplification of emerging viruses in a bat colony. Emerg. doi:10.1073/pnas.0506735102 Infect. Dis. 17,449–456. doi:10.3201/eid1703.100526 7. Memish, Z.A. et al. (2013) Middle East respiratory syndrome coronavirus in bats, 32. O’Shea, T.J. et al. (2014) Bat flight and zoonotic viruses. Emerg. Infect. Dis. 20, Saudi Arabia. Emerg. Infect. Dis. 19, 1819–1823. doi:10.3201/eid1911.131172 741–745. doi:10.3201/eid2005.130539 8. Góes, L.G.B. et al. (2016) Genetic diversity of bats coronaviruses in the Atlantic 33. Zhang, G.J. et al. (2013) Comparative Analysis of bat genomes provides insight Forest hotspot biome, Brazil. Infect. Genet. Evol. 44,510–513. doi:10.1016/ into the evolution of flight and immunity. Science 339,456–460. doi:10.1126/ j.meegid.2016.07.034 science.1230835 9. Spaan, W.J.M. et al. (2005) Virus . Fauquet, C.M. et al., eds. San Diego: 34. Roberts, B.J. et al. (2012) Long-distance and frequent movements of the flying-fox Elsevier Inc. Pteropus poliocephalus: implications for management. PLoS One 7, e42532. 10. Lai, M.M. and Cavanagh, D. (1997) The molecular biology of coronaviruses. Adv. doi:10.1371/journal.pone.0042532 Virus Res. 48,1–100. doi:10.1016/S0065-3527(08)60286-9 35. Breed, A.C. et al. (2010) Bats without borders: long-distance movements and 11. Fraenkel-Conrat, H. et al. (1988) Virology. New Jersey: Prentice-Hall. implications for disease risk management. EcoHealth 7, 204–212. doi:10.1007/ s10393-010-0332-z 12. Hartmann, K. (2005) Feline infectious peritonitis. Vet. Clin. North Am. Small Anim. Pract. 35, 39. doi:10.1016/j.cvsm.2004.10.011 36. Jeong, J. et al. (2017) Persistent infections support maintenance of a coronavirus in a population of Australian Epidemiol. Infect. (in press). 13. Weiss, S.R. and Navas-Martin, S. (2005) Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol. Mol. Biol. 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He is now a scientist with Biosecurity Queensland, different strains of severe acute respiratory syndrome-related Rhinolophus bat Department of Agriculture and Fisheries, where he recently man- coronavirus in China reveal bats as a reservoir for acute, self-limiting infection that allows recombination events. J. Virol. 84, 2808–2819. doi:10.1128/JVI.02219-09 aged projects for the National Hendra Virus Research Program.

MICROBIOLOGY AUSTRALIA * MARCH 2017 11 In Focus

Filoviruses and bats

Amy J Schuh A , Brian R Amman A and Jonathan S Towner A,B AViral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA BTel: +1 404 639 4561, Fax: +1 404 639 1509, Email: [email protected]

While Reston and Lloviu viruses have never been associated among macaques exported to the United States from the Philip- with human disease, the other filoviruses cause outbreaks pines5.Taœ Forest virus has been isolated once only from a nonfatal of hemorrhagic fever characterised by person-to-person case that became ill following the necropsy of a chimpanzee transmission and high case fatality ratios. Cumulative that died from a hemorrhagic disease in Côte d’Ivoire in 19946. evidence suggests that bats are the most likely reservoir Bundibugyo virus was initially isolated during a FHF outbreak hosts of the filoviruses. Ecological investigations following in Uganda in 20077. Lloviu virus was identified during the investi- outbreaks associated with entry gation of a die-off of Miniopterus schreibersii bats in Spain in into caves inhabited by Rousettus aegyptiacus bats led to 20028. A partial genomic sequence recovered from a Rousettus the identification of this bat species as the leschenaultii bat captured in China in 2013 likely represents a novel host of the . Experimental infection of filovirus9. Ecological niche modelling has confirmed the known R. aegyptiacus with Marburg virus has provided insight range of filovirus circulation and has predicted additional areas into the natural history of filovirus infection in bats that throughout sub-Saharan Africa and Southeast Asia that are suitable – may help guide the search for the reservoir hosts of the for zoonotic transmission of filoviruses10 13. . Evidence suggests that bats are natural reservoir Filovirus history and geographic range hosts of the filoviruses The phylogeny illustrates the genetic relationships between the Although contact with non-human or duiker tissue has – filoviruses and the associated map shows the known range of been linked to FHF outbreaks1,14 16, the high mortality caused by filovirus circulation according to virus (Figure 1). Marburg virus filoviruses in these animals indicate that they are only incidental (MARV) was first described in 1967 following two successive filo- hosts. However, FHF outbreak investigations have revealed that virus hemorrhagic fever (FHF) outbreaks among German and many of the index cases had entered environments inhabited by former-Yugoslavian laboratory workers that had handled bats prior to disease onset. In 1975, MARV disease occurred in a imported from Uganda1. Ravn virus (RAVV), also a , tourist that had stayed in two hotels populated with bats and visited was initially isolated from a 1987 fatal case in Kenya2. Nearly Chinhoyi Caves in present-day Zimbabwe 8–9 days prior to disease simultaneous FHF outbreaks in present-day South Sudan and the onset17. The index case in the 1976 outbreak of SUDV disease Democratic Republic of the Congo (DRC), led to the identification worked at a cotton factory containing Mops trevori18 and the index of Sudan virus (SUDV)3 and Ebola virus (EBOV)4, respectively. case in the 1979 SUDV disease outbreak worked at the same was discovered in 1989 following an epizootic of FHF factory19. Fifteen days before becoming ill, the index case in the

12 10.1071/MA17005 MICROBIOLOGY AUSTRALIA * MARCH 2017 In Focus

Isolate (year) Virus Species Genus

01 DRC (1999)

100 Ozolins (1975)

Marburg Popp (1967) 91 virus

69 Musoke (1967) 50 Marburgvirus Marburg Ang 0126 (2005) 100 marburgvirus

Ravn (1987)

Ravn 99 188 Bat (2007) virus 64 09 DRC (1999)

BtFV/WD04 (2013)

Lloviu Lloviu MS-Liver-86 (2013) Cuevavirus virus cuevavirus

100 Makona (2014) Ebola Zaire 100 Mayinga (1976) virus 59 100 807460 (1994) 100 Taï Forest Taï Forest Taï Forest (1994) virus ebolavirus

100 Bundibugyo (2007) Ebolavirus Bundibugyo Bundibugyo 100 virus ebolavirus 99 EboBund-112 (2012)

Gulu (2000) Sudan Sudan 100 virus ebolavirus Boneface (1976) 94 Reston Reston Ferlite (1999) virus ebolavirus 0.1

Angola Gabon South Sudan

China Guinea Philippines Uganda

DRC Côte d'Ivoire Spain Zimbabwe

Figure 1. Filovirus maximum-likelihood phylogeny and geographic distribution. The phylogeny was derived from concatenated partial nucleoprotein, 35 and RNA-dependent RNA polymerase filovirus gene sequences. A single representative sequence from each country in which filovirus zoonotic spillover has been detected or spillover into humans has occurred was selected to capture the geographic range of virus circulation. Sequences are coloured according to the sampling location and the colours correspond to those used in the associated map and legend. The numbers to the lower-left of the nodes are bootstrap percentages based on 1000 replicates. Horizontal branch lengths are proportional to the genetic distance between sequences and the scale underneath the phylogeny indicates the number of nucleotide substitutions per site.

1980 MARV disease outbreak had entered Kenya’s bat-populated inoculated with EBOV21. Three bat species (Mops condylurus, Elgon Caves2 and the 1987-isolated case of RAVV disease had visited Chaerephon pumilus and wahlbergi) supported Kenya’s Kitum Cave prior to becoming ill20. After the large 1995 EBOV replication and seroconverted in the absence of overt clinical epidemic of EBOV disease in present-day DRC, 24 and 19 disease, while the remaining animal and plant species were refrac- and invertebrate native species were experimentally tory to virus infection. These findings supported the accumulating

MICROBIOLOGY AUSTRALIA * MARCH 2017 13 In Focus

number of links between FHF index cases and prior exposure to mechanisms of bat-to-bat MARV transmission, a third study by environments inhabited by bats. This linkage became stronger Paweska et al. housed groups of donor bats inoculated with a when it was discovered that 52% of the 154 cases in a series of human MARV strain with naïve contact bats in direct, indirect or MARV disease outbreaks in the DRC between 1998 and 2000 airborne contact and monitored for evidence of infection for worked in the underground Goroumbwa Mine known to house 42 days30. No evidence of infection was detected in the contact hundreds of thousands of bats22. In 2007, an EBOV disease out- bats; however, the inoculated bats shed little to no MARV in their break followed a reported annual migration of Hypsignathus bodily fluids and were serially sacrificed as the study progressed. monstrosus and franqueti and the putative index case The possibility that hematophagous ectoparasitic argasid had purchased bats for consumption23. The index cases in a series (Ornithodoros faini) found in large colonies of R. aegyptiacus of MARV and RAVV disease outbreaks in 2007 worked in Kitaka might facilitate marburgvirus transmission was ruled-out when Mine, Uganda24 and two cases of MARV disease were found in >3000 O. faini ticks collected from Python Cave tested negative tourists that had separately visited nearby-Python Cave in 200825,26. for marburgvirus RNA31. Further studies are needed to determine how MARV is maintained in its natural reservoir host. Rousettus aegyptiacus identified as a natural reservoir host for the marburgviruses Ecological investigations following the 2007–2008 MARV and RAVV Search for the natural reservoir hosts disease outbreaks in Uganda revealed that Kitaka Mine and Python of the ebolaviruses R. aegyptiacus24,27 Cave were inhabited by large numbers of . Although the index cases of ebolavirus disease outbreaks have been R. aegyptiacus Follow-up longitudinal studies of populations at linked to bats, they have never been associated with a particular thesesites revealed a consistentprevalenceof both MARV and RAVV environment, such as caves, like the index cases of marburgvirus – infection in 2 5% of the bats. Genetically diverse marburgviruses disease outbreaks. Therefore, the search for the reservoir hosts of were isolated from bat tissues that were genetically similar to those the ebolaviruses has involved testing a wide-range of wild-caught, sequences generated from outbreak cases. Further, the studies forest-dwelling bats for evidence of ebolavirus infection. Serolog- found a temporal association between marburgvirus spillover ical reactivity of bat sera with ebolavirus antigen has been detected events, biannual pulses of active MARV infection in juvenile bats in 307 bats representing at least 17 species throughout sub-Saharan and the biannual birthing season. These studies provided the Africa and Asia32–40. Evidence of active ebolavirus infection has fi R. aegyptiacus evidence needed to de nitively identify as a natural been found in seven bat species – EBOV RNA has been detected in reservoir host of the marburgviruses and a source of spillover into three solitary, forest-dwelling frugivorous species (E. franqueti, the human population. H. monstrosus and torquata) captured in Gabon and the Republic of Congo32 and RESTV RNA has been detected in four Natural history of MARV infection diverse species (Chaerephon plicatus, brachyotis, in R. aegyptiacus Miniopterus australis and M. schreibersii) captured in the Philip- Following the discovery of R. aegyptiacus as the natural reservoir pines39. However, infectious ebolavirus has never been isolated host for the marburgviruses, experimental studies were initiated to from any of these bat species. Consequently, it is unknown whether investigate the natural history of virus infection in this bat species. they are primary reservoir hosts of the virus, secondary reservoir The first published study by Paweska et al. found that bats inoc- hosts that play a minor role in virus maintenance or incidental dead- ulated by the intraperitoneal and subcutaneous routes with a Vero end hosts that are susceptible to infection, but do not shed cell-adapted, human-derived MARV strain exhibited viral replica- infectious virus. It is interesting to note that MARV RNA in the tion in multiple tissues in the absence of overt illness followed by absence of infectious virus has been detected in Miniopterus seroconversion, while bats dually inoculated by the oral and nasal inflatus, Rhinolophus eloquens and Hipposideros sp. bats that routes showed no evidence of infection within the 21-day study roost with R. aegyptiacus24,41. Similarly, investigations examining period28. A second study by Amman et al. found that bats subcu- the susceptibility of R. aegyptiacus bats to experimental infection taneously inoculated with a low-passage, bat-derived MARV strain with each of the five ebolaviruses demonstrated very limited shed virus in their oral secretions up to 11 days following infection replication and no viral shedding followed by seroconversion42,43. and led to the hypothesis that the virus may be horizontally These findings suggest that sporadic detection of filovirus RNA or transmitted between bats through direct and/or indirect contact IgG antibodies from wild-caught bats may only represent virus with infectious oral secretions or biting29. To investigate the spillover resulting from contact with a primary reservoir host.

14 MICROBIOLOGY AUSTRALIA * MARCH 2017 In Focus

Expectations of a filovirus natural reservoir host 6. Formenty, P. et al. (1999) Human infection due to Ebola virus, subtype Cote d’Ivoire: clinical and biologic presentation. J. Infect. Dis. 179,S48–S53. Based on what we have learned about marburgvirus infection in doi:10.1086/514285 R. aegyptiacus, we would expect the reservoir hosts of the ebola- 7. Towner, J.S. et al. (2008) Newly discovered Ebola virus associated with hemor- rhagic fever outbreak in Uganda. PLoS Pathog. 4, e1000212. doi:10.1371/journal. viruses to have a consistent prevalence of both active and past ppat.1000212 infection, shed sufficiently high levels of infectious virus to maintain 8. Negredo, A. et al. (2011) Discovery of an ebolavirus-like filovirus in Europe. PLoS Pathog. 7 virus circulation in the population and exhibit host population , e1002304. doi:10.1371/journal.ppat.1002304 9. He, B. et al. (2015) Filovirus RNA in fruit bats, China. Emerg. Infect. Dis. 21, dynamics conducive to virus transmission. Host population-level 1675–1677. doi:10.3201/eid2109.150260 virus persistence is highly dependent on host population dynamics, 10. Peterson, A.T. et al. (2004) Ecologic and geographic distribution of filovirus particularly community size and annual fluctuations in age-struc- disease. Emerg. Infect. Dis. 10,40–47. doi:10.3201/eid1001.030125 11. Peterson, A.T. et al. (2006) Geographic potential for outbreaks of Marburg ture from births and deaths. Mathematical modelling of marburg- hemorrhagic fever. Am. J. Trop. Med. Hyg. 75,9–15. R. aegyptiacus virus transmission in a closed population of revealed 12. Pigott, D.M. et al. (2014) Mapping the zoonotic niche of Ebola virus disease in that the virus was only able to persist if the model included: (1) a Africa. eLife 3, e04395. doi:10.7554/eLife.04395 et al biannual breeding component that provided a twice-yearly influx of 13. Pigott, D.M. . (2015) Mapping the zoonotic niche of Marburg virus disease in Africa. Trans. R. Soc. Trop. Med. Hyg. 109, 366–378. doi:10.1093/trstmh/trv024  susceptible juveniles; (2) a latent period of 21 days; and (3) a host 14. Le Guenno, B. et al. (1995) Isolation and partial characterisation of a new strain of population size 20 00044. This suggests that if the natural reser- Ebola virus. Lancet 345, 1271–1274. doi:10.1016/S0140-6736(95)90925-7 et al voirs of the ebolaviruses are a solitary bat species that only con- 15. Leroy, E.M. . (2004) Multiple Ebola virus transmission events and rapid decline of central African wildlife. Science 303, 387–390. doi:10.1126/science. gregates during the breeding season(s), host population-level virus 1092528 maintenance may depend on other mechanisms such as persistent 16. Georges, A.J. et al. (1999) Ebola hemorrhagic fever outbreaks in Gabon, 1994- –1997: epidemiologic and health control issues. J. Infect. Dis. 179,S65–S75. infection with intermittent shedding, as has been observed with doi:10.1086/514290 45–49 other bat-borne viruses . Thelarge number of batspecies within 17. Conrad, J.L. et al. (1978) Epidemiologic investigation of Marburg virus disease, the geographical range of ebolavirus circulation complicates the Southern Africa, 1975. Am. J. Trop. Med. Hyg. 27, 1210–1215. et al search for the natural reservoir host of these viruses. In an effort to 18. Arata, A.A. . (1978) Approaches towards studies on potential reservoirs of viral haemorrhagic fever in southern Sudan (1977). In Ebola virus haemorrhagic guide field sampling efforts, Peterson et al. used a series of fever (Pattyn, S.R., ed), pp. 191–202, Elsevier/Netherland Biomedical. biological principles to develop a priority list of mammalian clades 19. Baron, R.C. et al. (1983) Ebola virus disease in southern Sudan: hospital dissem- ination and intrafamilial spread. Bull. World Health Organ. 61,997–1003. that coincided with past filovirus disease outbreaks50 and Han et al. 20. Johnson, E.D. et al. (1996) Characterization of a new Marburg virus isolated from used a machine learning algorithm to identify potential filovirus- a 1987 fatal case in Kenya. Arch. Virol. Suppl. 11, 101–114. positive bat species based on intrinsic trait similarity with known 21. Swanepoel, R. et al. (1996) Experimental inoculation of and animals with Ebola virus. Emerg. Infect. Dis. 2, 321–325. doi:10.3201/eid0204.960407 filovirus RNA-, isolation- and antibody- positive bat species51. 22. Bausch, D.G. et al. (2006) Marburg hemorrhagic fever associated with multiple genetic lineages of virus. N. Engl. J. Med. 355,909–919. doi:10.1056/NEJMoa For more information on filoviruses and bats, we would like to 051465 direct readers to recent overviews published by Olival and Hay- 23. Leroy, E.M. et al. (2009) Human Ebola outbreak resulting from direct exposure to man52, Wood et al.53, Leendertz et al.54 and Amman et al55. fruit bats in Luebo, Democratic Republic of Congo, 2007. Vector Borne Zoonotic Dis. 9,723–728. doi:10.1089/vbz.2008.0167 24. Towner, J.S. et al. (2009) Isolation of genetically diverse Marburg viruses Acknowledgement from Egyptian fruit bats. PLoS Pathog. 5, e1000536. doi:10.1371/journal.ppat. 1000536 fi The ndings and conclusions in this report are those of the authors 25. Timen, A. et al. (2009) Response to imported case of Marburg hemorrhagic and do not necessarily represent the official position of the Centers fever, the Netherlands. Emerg. Infect. Dis. 15, 1171–1175. doi:10.3201/eid1508. 090015 for Disease Control and Prevention. 26. Centers for Disease Control and Prevention. (2009) Imported case of Marburg hemorrhagic fever – Colorado, 2008. Morb. Mortal. Weekly Rep. 58, 1377–1381. References 27. Amman, B.R. et al. (2012) Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human Primate 1. Smith, M.W. (1982) Field aspects of the Marburg virus outbreak: 1967. infection. PLoS Pathog. 8, e1002877. doi:10.1371/journal.ppat.1002877 Supply 7,11–15. 28. Paweska, J.T. et al. (2012) Virological and serological findings in Rousettus et al Lancet 319 – 2. Smith, D.H. . (1982) Marburg-virus disease in Kenya. ,816 820. aegyptiacus experimentally inoculated with Vero cells-adapted Hogan strain of doi:10.1016/S0140-6736(82)91871-2 Marburg virus. PLoS One 7, e45479. doi:10.1371/journal.pone.0045479 3. Report of a WHO/International Study Team. (1978) Ebola haemorrhagic fever in 29. Amman, B.R. et al. (2015) Oral shedding of Marburg virus in experimentally Bull. World Health Organ. 56 – Sudan, 1976. , 247 270. infected Egyptian fruit bats (Rousettus aegyptiacus). J. Wildl. Dis. 51,113–124. 4. Reportof an InternationalCommission. (1978) Ebola haemorrhagic fever inZaire, doi:10.7589/2014-08-198 Bull. World Health Organ. 56 – 1976. , 271 293. 30. Paweska, J.T. et al. (2015) Lack of Marburg virus transmission from experimentally 5. Miranda, M.E. et al. (1999) Epidemiology of Ebola (subtype Reston) virus in the infected to susceptible in-contact Egyptian fruit bats. J. Infect. Dis. 212,S109– Philippines, 1996. J. Infect. Dis. 179, S115–S119. doi:10.1086/514314 S118. doi:10.1093/infdis/jiv132

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31. Schuh, A.J. et al. (2016) No evidence for the involvement of the argasid 46. Baer, G.M. et al. (1966) Bat salivary gland virus carrier state in a naturally infected Ornithodoros faini in the enzootic maintenance of marburgvirus within Egyptian Mexican freetail bat. Am. J. Trop. Med. Hyg. 15,769–771. Rousettus aegyptiacus. Parasit. Vectors 9 rousette bats , 128. doi:10.1186/ 47. Lumsden, W.H. et al. (1961) A virus from insectivorous bats in Uganda. Ann. Trop. s13071-016-1390-z Med. Parasitol. 55, 389–397. doi:10.1080/00034983.1961.11686063 et al Nature 438 32. Leroy, E.M. . (2005) Fruit bats as reservoirs of Ebola virus. , 48. Bell, J.F. et al. (1964) A new virus, ‘MML’, enzootic in bats (Myotis lucifugus)of – 575 576. doi:10.1038/438575a Montana. Am. J. Trop. Med. Hyg. 13,607–612. et al 33. Pourrut, X. . (2007) Spatial and temporal patterns of antibody 49. Sulkin, S.E. et al. (1959) Studies on the pathogenesis of rabies in insectivorous J. Infect. Dis. 196 – prevalence in the possible reservoir bat species. , S176 S183. bats. I. Role of brown adipose tissue. J. Exp. Med. 110,369–388. doi:10.1084/ doi:10.1086/520541 jem.110.3.369 et al 34. Pourrut, X. . (2009) Large serological survey showing cocirculation of Ebola 50. Peterson, A.T. et al. (2004) Potential mammalian filovirus reservoirs. Emerg. and Marburg viruses in Gabonese bat populations, and a high seroprevalence of Infect. Dis. 10, 2073–2081. doi:10.3201/eid1012.040346 both viruses in Rousettus aegyptiacus. BMC Infect. Dis. 9, 159. doi:10.1186/ 51. Han, B.A. et al. (2016) Undiscovered bat hosts of filoviruses. PLoS Negl. Trop. Dis. 1471-2334-9-159 10, e0004815. doi:10.1371/journal.pntd.0004815 35. Hayman, D.T. et al. (2010) Long-term survival of an urban fruit bat seropositive 52. Olival, K.J. and Hayman, D.T.S. (2014) Filoviruses in bats: current knowledge and for Ebola and Lagos bat viruses. PLoS One 5, e11978. doi:10.1371/journal. future directions. Viruses 6,1759–1788. doi:10.3390/v6041759 pone.0011978 53. Wood, J.L. et al. (2016) Ebola, bats and evidence-based policy: informing Ebola 36. Hayman, D.T. et al. (2012) Ebola virus antibodies in fruit bats, Ghana, West Africa. policy. EcoHealth 13,9–11. doi:10.1007/s10393-015-1050-3 Emerg. Infect. Dis. 18, 1207–1209. doi:10.3201/eid1807.111654 54. Leendertz, S.A. et al. (2016) Assessing the evidence supporting fruit bats as 37. Yuan, J. et al. (2012) Serological evidence of ebolavirus infection in bats, China. the primary reservoirs for Ebola viruses. EcoHealth 13,18–25. doi:10.1007/ Virol. J. 9, 236. doi:10.1186/1743-422X-9-236 s10393-015-1053-0 38. Olival, K.J. et al. (2013) Ebola virus antibodies in fruit bats, Bangladesh. Emerg. 55. Amman, B.R. et al. (In press) Ecology of filoviruses. In Marburg and Ebola Infect. Dis. 19,270–273. doi:10.3201/eid1902.120524 viruses: from ecosystems to molecules (Muehlberger, E. et al., eds). Current et al 39. Jayme, S.I. . (2015) Molecular evidence of Ebola Reston virus infection in Topics in Microbiology and Immunology, Springer. Philippine bats. Virol. J. 12, 107. doi:10.1186/s12985-015-0331-3 40. Ogawa, H. et al. (2015) Seroepidemiological prevalence of multiple species of filoviruses in fruit bats (Eidolon helvum) migrating in Africa. J. Infect. Dis. 212, S101–S108. doi:10.1093/infdis/jiv063 Biographies 41. Swanepoel, R. et al. (2007) Studies of reservoir hosts for Marburg virus. Emerg. Amy Schuh Brian Amman Infect. Dis. 13, 1847–1851. doi:10.3201/eid1312.071115 , PhD is a microbiologist, , PhD is an 42. Jones, M.E. et al. (2015) Experimental inoculation of Egyptian rousette bats ecologist and Jonathan Towner, PhD is the Team Lead of the Rousettus aegyptiacus Ebolavirus Marburgvirus ( ) with viruses of the and Virus-Host Ecology Section at the Viral Special Pathogens Branch at genera. Viruses 7, 3420–3442. doi:10.3390/v7072779 43. Paweska, J.T. et al. (2016) Experimental inoculation of Egyptian fruit bats the United States Centers for Disease Control and Prevention. They (Rousettus aegyptiacus) with Ebola virus. Viruses 8, 29. doi:10.3390/v8020029 conduct ecological investigations aimed at identifying the reservoir 44. Hayman, D.T. (2015) Biannual birth pulses allow filoviruses to persist in bat hosts of the filoviruses and use captive-born R. aegyptiacus bats to populations. Proc. Biol. Sci. 282, 20142591. doi:10.1098/rspb.2014.2591 study the mechanisms of filovirus maintenance and virus spillover 45. Constantine, D.G. et al. (1964) Latent infection of Rio Bravo virus in salivary glands of bats. Public Health Rep. 79, 1033–1039. doi:10.2307/4592318 to humans.

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The impact of novel lyssavirus discovery

Anthony R Fooks Wildlife Zoonoses and Vector-borne Ashley C Banyard Diseases Research Group, Animal and Plant Health Agency (APHA) Wildlife Zoonoses and Vector-borne Weybridge, UK Diseases Research Group, Animal Institute for Infection and Immunity and Plant Health Agency (APHA) St George’s Hospital Medical School Weybridge, UK University of London, London, UK Tel: +44 (0) 20 8026 9463 Tel: +44 (0) 20 8415 2238 Email: [email protected] Email: [email protected]

The global discovery of novel is of continued populations, the association of bats with lyssaviruses is clear. scientific interest through its importance to both public and Historically, rabies has a long association with hematophagous bats animal health. Lyssaviruses cause an invariably fatal en- across Central and South America and this association, with cephalitis that is more commonly known as rabies. The ‘mysterious blood feeding creatures of the night’, has cemented term rabies has a long history in human society, as rabies rabies into the conscience of human populations. Alongside this virus (RABV) is the only pathogen that is associated with the more typical association of the virus with aggressive , the 100% fatality once the onset of clinical disease has started. horrific clinical disease seen in human infection, and the invariably Although predominantly associated across the globe with fatal nature of infection, has led to rabies being the most feared domestic and feral dog populations, the association of pathogen known to man. Such clinical manifestations have held the bats is clear. Whilst evolutionarily associated with bats, imagination of humanity since the earliest reports of canine and RABV is most commonly transmitted to human populations human madness. Whilst evolutionarily associated with bats, rabies through the bite of an infected dog and dogs are considered virus is most commonly transmitted to human populations through the primary reservoir of disease. Indeed, RABV does cause the bite of an infected dog. Indeed, RABV does cause thousands of more than an estimated 70 000 deaths every year globally deaths every year globally in human populations and whilst this is in human populations and whilst this is largely in areas largely in areas where the virus is endemic, areas that remain free of where the disease is endemic, areas that remain free of rabies must remain vigilant to the risk of re-incursion of disease2. rabies must remain vigilant to the risk of re-incursion of Alongside the burden seen from dogs, wildlife species also play an disease. Characterisation of novel lyssaviruses is of impor- important role in the epidemiology of disease although the paucity tance on several levels. Not least to investigate the patho- of data on wild animal populations, their distribution and the genesis and potential transmission routes of different generally sporadic interactions between different wildlife popula- lyssavirus species but also to assess the potential effect of tions and domesticated carnivore species means that the role of post-exposure treatments and should human wildlife and the epidemiology of the virus is often unclear. exposure occur. Bat lyssaviruses and the problems associ- Alongside RABV strains that are predominantly associated with ated with novel discoveries and the potential impact they terrestrial carnivore species, a number of other genetically and to have on both human and animal populations are discussed. some extent antigenically related viruses exist within the lyssavirus The global discovery of lyssaviruses is of continued scientific genus3. Currently, 14 viruses, in addition to RABV are associated interest through its importance to both public and animal health. with bats and are classified within the genus4, while few are well Lyssaviruses cause an invariably fatal encephalitis referred to as characterised from either an epidemiological or clinical disease rabies. The term rabies has a long history in human society, as rabies perspective. Three bat-associated viruses, Lleida bat lyssavirus, virus (RABV) is the only pathogen that is associated with 100% Gannoruwa bat lyssavirus and Taiwan bat lyssavirus, are not offi- fatality once the onset of clinical disease has started1. Whilst cially classified within the Lyssavirus genus, and remain as tentative predominantly associated across the globe with domestic and feral species only. Although RABV causes a significant annual disease

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17006 17 In Focus

Table 1. The association of lyssavirus species with bats. For species coloured green there appears to be no protection afforded by current rabies vaccines. IKOV and MOKV are absent as there has been no association with bat species. Continent Geographical Lyssavirus species Bat species associated Common bat name Human distribution of isolates with lyssavirus fatalities infection reported The Americas North and South America Rabies lyssavirus (RABV)A Eptesicus fuscus Big brown bat Yes Tadarida brasiliensis Mexican/Brazilian free-tail bat Lasionycteris noctivagens Silver-haired bat Perimyotis subflavus Tri-coloured bat Desmodus rotundus Vampire bat Africa Nigeria, Senegal, Ghana, (LBV) Eidolon helvum Straw coluored fruit bat No Kenya France (ex-Togo or Egypt), Rousettus aegyptiacus Kenya, Central African Republic pussilus Dwarf epaulet fruit bat Ghana Epomorphus giambianus Gambian epauletted fruit bat Ghana Epomops buettikoferi Buettikofer’s epauletted fruit bat Guinea Nycteris gambiensis Gambian slit-faced bat South Africa Epomorphorus wahlbergi Wahlberg’s epauletted fruit bat Kenya Shimoni bat lyssavirus Hipposideros commersoni Commerson’s leaf-nosed No (SHIBV) bat South Africa, KenyaB Miniopterus sp. Undefined Yes (DUVV) Zimbabwe Nycteris thebaica Egyptian slit-faced bat Europe France, Germany, Spain European bat 1 Lyssavirus Eptesicus serotinus Serotine bat Yes (EBLV-1) The Netherlands, European bat 2 lyssavirus Myotis daubentonii Daubenton’s bat Yes Switzerland, UK, France, (EBLV-2) Germany, Luxembourg, Finland Germany, France Bokeloh bat lyssavirus Myotis nattereri Natterer’s bat No (BBLV) Spain Lleida bat lyssavirus Miniopterus schreibersii Common bent-winged bat No (LLEBV)C Eurasia Kyrgystan Aravan lyssavirus (ARAV) Myotis blythi Lesser mouse-eared bat No Russian Federation, China Irkut lyssavirus (IRKV) Murina leucogaster Greater tube-nosed bat Yes Tajikistan Khujand lyssavirus (KHUV) Myotis mystacinus Whiskered bat No Russian Federation, KenyaD West Caucasian bat Miniopterus schreibersii Common bent-winged bat No lyssavirus (WCBV) Australasia Australia Australian bat lyssavirus Pteropus alecto Black flying fox and related Yes (ABLV) sp. Saccolaimus flaviventris Yellow-bellied sheath-tailed bat Asia Gannoruwa bat lyssavirus Pteropus medius Indian flying fox No (GBLV)C Taiwan Taiwan bat lyssavirus Pipistrellus abramus Japanese house bat No (TWBLV)C

AMore than 50 bat species have been implicated in RABV infection across the Americas- the most frequently reported species are listed for convenience. BCase reported from The Netherlands but origin of exposure was Kenya. CNot yet classified as lyssaviruses by the International Committee for the Taxonomy of Viruses (ICTV). DSerological evidence of infection in Kenya.

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burden to human populations globally, the association of these recent reports have highlighted the importance of seeking post- other lyssaviruses with human fatalities is poorly characterised exposure prophylaxis wherever interaction with a potentially rabid (Table 1). This may be because there are genuinely fewer cases bat has occurred, even in the absence of any actual contact between of infection with these viruses across human and animal popula- the animal and the human individual in question12. Interestingly tions or, conversely may reflect the inability of the most commonly however, the detection of classical RABV has never been reported used diagnostic tool, the antigen detection based Fluorescent across the ‘Old World’ in bats13. In contrast, the other viruses that Antibody Test (FAT), to differentiate between lyssavirus species are classified within the lyssavirus genus have, in the main, been and a lack of a secondary confirmatory test in endemic regions that isolated from different bat species across the Old World with a can genetically type virus5. This feature is important and as more complete lack of reporting of any lyssavirus species, apart from divergent lyssaviruses are discovered the ability of commercial RABV, in the Americas (Figure 1). The basis for this apparent conjugates to detect them requires assessment. These factors mean division remains unknown. It is probable that RABV has existed in that the true epidemiological situation regarding these lyssaviruses blood-sucking and insectivorous bats in the Americas for millennia. remains unclear and consequently the burden of disease to animal With the introduction of European dog rabies and large agricultural and human health remains undefined6. hosts (, horses etc.) following the European colonisation of the Americas, the incidence of RABV in these new hosts likely Divergent lyssaviruses have been recognised for over 65 years. increased exponentially. Further, increased agricultural prey spe- Initial discoveries of viruses causing rabies disease originated in the cies likely impacted significantly to the increase in vampire bat fi Old World in the 1950s where serological pro ling using mono- populations. clonal antibodies characterised viruses similar to RABV7. A steady fi flow of virus discovery then continued until molecular methods A key factor that de nes the relationship between viruses and were developed that superseded antibody based classification of their hosts is the ability to be transmitted and maintained within a new pathogens. Currently, sequencing technologies are able to population. The maintenance of lyssaviruses within bat popula- fi rapidly type viruses genetically and as such lyssaviruses continue to tions is poorly understood. Whilst bats can be clearly identi ed as be discovered8,9. Apart from those already detected and partially reservoir hosts for lyssaviruses, where associations have been fi fi characterised, novel species continue to be detected that then de ned, they do not ful l the classic ideal of a symbiotic relation- require characterisation and classification. The importance of novel ship between microbe and host. In this sense, the ability of viruses lyssaviruses remains unknown but the potential for fatal infection to cause clinical disease is of interest. For numerous other patho- following spill over events dictates that some importance must be gens for which bats are considered reservoirs, including high fi placed on their virological characterisation. This has become pro le viral zoonotic pathogens such as Ebola virus and Nipah increasingly important with the heightened interest in bats as virus, infection generally occurs in the absence of clinical disease biological entities and as reservoirs of zoonotic pathogens that in the bat. For lyssaviruses, although our understanding may be have crossed species barriers to drive large mortality events in limited, this feature of virus host interactions is not true. Indeed, human and animal populations. Furthermore, the potential for lyssaviruses do cause clinical disease in the bats they infect and interactions with bat species has increased with the encroachment in the vast majority of cases it is only through the observation of of human populations into areas of forestation and the popularity of clinical manifestations of disease that these viruses are discovered. human activities such as caving and potholing10. This is a feature not restricted to bat infection, but one that covers lyssavirus infection of all mammals. However, recent evidence The association of lyssaviruses with bats provides several interest- has suggested that mammals can be exposed to virus, mount an ing conundrums regarding the epidemiology of these viruses11. immunologically detectable response (generally a humoral re- RABV is present across terrestrial carnivore populations across the sponse) and clear the virus. Both in bat populations where sero- globe but is only associated with the infection of insectivorous, logical positivity can range significantly across a roost and human hematophagous and to a lesser extent frugivorous bats across the populations, evidence of exposure in the absence of clinical disease Americas. In the Americas, following the successful elimination of has been reported. What however constitutes exposure against an rabies from domestic carnivore species and, following extensive interaction that leads to productive infection and disease is ill oral vaccination programs, reduction of disease within wildlife defined, as are the mechanisms that dictate the outcome of any populations, bats represent an ever present remaining source potential exposure/infection event. Assessment of innate signalling of virus for potential infection and spill over transmission events mechanisms in bats, and other species, following exposure needs to that continue to cause fatalities in the human populations. Indeed, be completed to understand the mechanisms that drive infection.

MICROBIOLOGY AUSTRALIA * MARCH 2017 19 In Focus

Another key question when considering bat lyssavirus infection is threshold will protect individuals from the development of dis- the potential for cross species transmission events (CSTs)14. Whilst ease17. However, the cut-off for a serological neutralising antibody few CSTs have been reported where the bat variant has been titre is poorly defined for numerous viruses within the continually maintained in terrestrial hosts15,16, the potential for such events expanding lyssavirus genus and as such the discovery of novel remains. Certainly across the Old World, where non-rabies lyssa- viruses requires investigation as to the efficacy of existing pre- and – viruses appear to predominate in bat populations whilst RABV post-exposure preparations6,18 23. Of the more divergent viruses, appears to circulate terrestrially, such events may occur more often there appears to be no vaccine afforded protection whilst the less but without any evidence for viral adaptation and transmission. divergent may be neutralised by a vaccinal response5,6,24,25. Current diagnostic methods to genetically type at post The relative impact of these viruses on human populations is low, mortem cannot differentiate between lyssaviruses and as such CST as evidenced by the low number of human fatalities associated events may be missed. Certainly, without the adoption of molecular with these viruses in countries that are free of terrestrial rabies tools to genetically type circulating virus variants such events will where diagnostic capabilities are able to thoroughly investigate remain undefined. cases of encephalitis. Maintenance of such rabies free areas is A final element of importance when considering the detection of important and the OIE definition of what constitutes freedom novel lyssaviruses, and their effect on human and animal popula- from rabies disease is clear and valid. Numerous island nations, tions, is the role of neutralising antibodies. Whilst nearly 100% including the United Kingdom and Australia, are defined by the fatal following the development of clinical disease, it has long OIE as ‘rabies free’, because rabies virus is absent in terrestrial been established that a neutralising antibody titre over a defined mammals. However, both serological and virological evidence for

European bat 1 lyssavirus Spill-over into European bat 2 terrestrial lyssavirus carnivores Australian bat lyssavirus

Duvenhage lyssavirus Irkut lyssavirus* Khujand lyssavirus* I ‘New ‘Old Human World’ World’ Taiwan bat infection Lyssavirus* Rabies Gannoruwa bat lyssavirus I lyssavirus* Bokeloh bat lyssavirus Aravan lyssavirus* II Shimoni bat lyssavirus*

Lagos bat lyssavirus III Mokola Vampire bat lyssavirus? infection of livestock Ikoma lyssavirus?* West Lleida bat Caucasian lyssavirus* bat lyssavirus*

Figure 1. The global diversity of the bat lyssaviruses. Where viruses have been associated with human fatalities the species is flanked by a human silhouette. The division of lyssavirus species into antigenically distinct phylogroups is shown as roman numerals within stars. *Denotes where only a single isolate exists. Lyssavirus species shown as black boxes have not been reported in bat species.

20 MICROBIOLOGY AUSTRALIA * MARCH 2017 In Focus

lyssaviruses continues to increase across bat populations. As such, 17. Warrell, M.J. (2012) Current rabies vaccines and prophylaxis schedules: prevent- ing rabies before and after exposure. Travel Med. Infect. Dis. 10,1–15. an increased knowledge of the mechanisms behind how these doi:10.1016/j.tmaid.2011.12.005 viruses persist, and how they cross the species barrier will be critical 18. Brookes, S.M. et al. (2006) Ability of rabies vaccine strains to elicit cross- neutralising antibodies. Dev. Biol. (Basel) 125,185–193. in ensuring that risk assessments surrounding human and animal 19. Brookes, S.M. et al. (2005) Rabies human diploid cell vaccine elicits cross- health are optimised. neutralising and cross-protecting immune responses against European and Australian bat lyssaviruses. Vaccine 23, 4101–4109. doi:10.1016/j.vaccine.2005. Acknowledgements 03.037 20. Hanlon, C.A. et al. (2005) Efficacy of rabies biologics against new lyssaviruses from ACB and ARF were jointly supported by the UK Department for Eurasia. Virus Res. 111,44–54. doi:10.1016/j.virusres.2005.03.009 et al Environment, Food and Rural Affairs (Defra), Scottish Government 21. Horton, D.L. . (2010) Quantifying antigenic relationships among the Lyssa- viruses. J. Virol. 84, 11841–11848. doi:10.1128/JVI.01153-10 and Welsh Government under project grant SV3500. 22. Nolden, T. et al. (2014) Comparative studies on the genetic, antigenic and pathogenic characteristics of Bokeloh bat lyssavirus. J. Gen. Virol. 95, 1647–1653. References doi:10.1099/vir.0.065953-0 23. Badrane, H. et al. (2001) Evidence of two Lyssavirus phylogroups with distinct et al 1. Fooks, A.R. . (2014) Current status of rabies and prospects for elimination. pathogenicity and immunogenicity. J. Virol. 75, 3268–3276. doi:10.1128/JVI.75. Lancet 384 – , 1389 1399. doi:10.1016/S0140-6736(13)62707-5 7.3268-3276.2001 et al 2. Banyard, A.C. . (2010) Reassessing the risk from rabies: a continuing threat 24. Horton, D.L. et al. (2014) Antigenic and genetic characterization of a divergent Virus Res. 152 – to the UK? ,79 84. doi:10.1016/j.virusres.2010.06.007 African virus, Ikoma lyssavirus. J. Gen. Virol. 95, 1025–1032. doi:10.1099/vir. 3. Baer, G.M. (1994) Rabies–an historical perspective. Infect. Agents Dis. 3,168–180. 0.061952-0 4. Afonso, C.L. et al. (2016) Taxonomy of the order Mononegavirales: update 2016. 25. Kuzmin, I.V. et al. (2008) Possible emergence of West Caucasian bat virus in Africa. Arch. Virol. 161, 2351–2360. doi:10.1007/s00705-016-2880-1 Emerg. Infect. Dis. 14,1887–1889. doi:10.3201/eid1412.080750 5. Fooks, A. (2004) The challenge of new and emerging lyssaviruses. Expert Rev. Vaccines 3, 333–336. doi:10.1586/14760584.3.4.333 6. Evans, J.S. et al. (2012) Rabies virus vaccines: is there a need for a -lyssavirus Biographies vaccine? Vaccine 30, 7447–7454. doi:10.1016/j.vaccine.2012.10.015 7. King, A.A. et al. (2004) Lyssavirus infections in European Bats. In: King, A.A., et al., Dr Banyard is a senior researcher in the Wildlife Zoonoses and eds. Historical perspective of Rabies in Europe and the Mediterranean basin. Vector-Borne Diseases Research Group at the Animal and Plant Paris, France: OIE. pp. 221–241. 8. Aréchiga Ceballos, N. et al. (2013) Novel lyssavirus in bat, Spain. Emerg. Infect. Health Agency (National Reference Laboratory for rabies). He has Dis. 19,793–795. doi:10.3201/eid1905.121071 20 years’ experience working with emerging veterinary pathogens 9. Gunawardena, P.S. et al. (2016) Lyssavirus in Indian Flying Foxes, Sri Lanka. and zoonoses. Dr Banyard’s research interests include molecular Emerg. Infect. Dis. 22, 1456–1459. doi:10.3201/eid2208.151986 pathogenesis of viral infections, novel pathogen discovery and 10. Engering, A. et al. (2013) Pathogen-host-environment interplay and disease emergence. Emerg. Microbes Infect. 2, e5. doi:10.1038/emi.2013.5 alternative approaches to vaccine development. 11. Badrane, H. and Tordo, N. (2001) Host switching in Lyssavirus history from the Chiroptera to the Carnivora orders. J. Virol. 75, 8096–8104. doi:10.1128/JVI.75. Dr Fooks leads the Wildlife Zoonoses and Vector-Borne Diseases 17.8096-8104.2001 Research Group at the Animal and Plant Health Agency. Since 12. Dato, V.M. et al. (2016) A systematic review of human bat rabies virus variant cases: evaluating unprotected physical contact with claws and teeth in support of 2002, he was appointed director of a World Health Organization accurate risk assessments. PLoS One 11, e0159443. doi:10.1371/journal.pone. Communicable Disease Surveillance and Response Collaborating 0159443 Centre for the characterisation of rabies and rabies-related viruses. 13. Rupprecht, C.E. et al. (2008) Can rabies be eradicated? Dev. Biol. (Basel) 131, 95–121. In 2006, he was appointed as an OIE Reference Expert for Rabies.He 14. Streicker, D.G. et al. (2010) Host phylogeny constrains cross-species emergence holds Honorary Visiting Professor positions in the Department of and establishment of rabies virus in bats. Science 329, 676–679. doi:10.1126/ Veterinary Pathology at the University of Liverpool, UK and in the science.1188836 15. Leslie, M.J. et al. (2006) Bat-associated rabies virus in skunks. Emerg. Infect. Dis. Institute for Infection and Immunity at St Georges Medical School, 12, 1274–1277. doi:10.3201/eid1208.051526 University of London, UK. Dr Fooks’ research interests are focused et al 16. Daoust, P.Y. . (1996) Cluster of rabies cases of probable bat origin among red on RNA viruses, especially viral diseases of the CNS and emerging/ foxes in Prince Edward Island, Canada. J. Wildl. Dis. 32,403–406. doi:10.7589/ 0090-3558-32.2.403 exotic viral zoonoses.

Microbiology Australia special issue: Breaking Research of Early Career and student Researchers

Early career (less than 5 year’s post-graduation) and student researchers who would like their area of research to be featured in Microbiology Australia are invited to contribute a proposal of their articles and its impact.

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MICROBIOLOGY AUSTRALIA * MARCH 2017 21 Under the Microscope

Menangle virus: one of the first of the novel viruses from fruit bats

Virus discovery and characterisation Attempts to isolate virus in a range of cell cultures was initially unsuccessful, but when tissues from piglets with severe defects Peter D Kirkland were examined, a number of isolates of a novel virus were Elizabeth Macarthur Agriculture obtained3. Virus was isolated most frequently from brain, lung and Institute Woodbridge Road heart and replicated well in BHK21 and HmLu-1 cells. Cytopathol- Menangle, NSW, Australia ogy, consisting of vacuolation of cells and large syncitia formation, Tel: +61 2 4640 6331 Fax: +61 2 4640 6429 was not usually observed until three passages in cell culture. A novel Email: [email protected] paramyxovirus, later named Menangle virus (MenV), was identified by electron microscopy. Subsequent molecular characterisation4 confirmed that this was a novel virus and its genome most closely aligned with those of viruses in the Rubulavirus genus, which includes human parainfluenza viruses 2 and 4 and virus. ‘Brainless pig disease swoops on Sydney.’ This was a media headline that threatened to emerge during the early stages of a disease outbreak in pigs in NSW. However, identifica- tion of the viral cause and epidemiological studies that supported a sound management program minimised the impact of this outbreak on animal and human health.

Disease outbreak and pathology In autumn 1997, an outbreak of severe reproductive failure oc- curred in a 2600-sow intensive piggery near Sydney, New South Wales1. Initially there was a high incidence of stillborn piglets and the delivery of mummified foetuses. Foetal deaths, considered to probably be associated with an in-utero infection, continued for a period of five months. Towards the end of the outbreak, many stillborn piglets with a range of severe defects were delivered (Figure 1). These congenital abnormalities mainly involved the central nervous system (reduced size of cerebellum, cerebral hemispheres, brain stem and spinal cord; hydranencephaly) and the skeletal system (arthrogryposis of the limbs; craniofacial deformities; scoliosis/kyphosis)2. Histologically there was a non- suppurative multifocal , and occasionally and . Intranuclear and intracytoplasmic were observed in neurons of the cerebrum and spinal cord. At the peak of the outbreak almost half of litters and most of the piglets in them were affected. The progression of the outbreak through different sections of the farm complex and the associated gross and microscopic pathological changes in affected piglets were Figure 1. Mummified and deformed piglets following infection with consistent with a viral infection. Menangle virus early in gestation.

22 10.1071/MA17007 MICROBIOLOGY AUSTRALIA * MARCH 2017 Under the Microscope

Diagnosis and epidemiology foxes7 while 6.5% of 5000 urine samples collected from flying boxes at locations along the coast of NSW between 2012 and 2015 gave Isolation of the causative viral agent allowed the development of positive qRT-PCR results (K Wernike and PD Kirkland, unpubl. a virus neutralisation test. Archival sera gave negative results, data). Collectively these data suggest that Menangle virus is demonstrating that the virus had recently entered the population5. endemic in Australian flying fox populations while there is sero- In contrast, positive results were obtained for serum from sows logical evidence to suggest that MenV is also present in Papua giving birth to infected piglets. Piglets born during the early stages New Guinea8. of the outbreak, and presumptively infected late in gestation when they are able to produce an immune response, also had positive serological results, which may explain the initial failure to isolate Eradication of the virus from pigs MenV. Usinga virusneutralisation test to test collectionsof archived Epidemiological studies5 had established that there were high sera, it was possible to monitor the spread of this virus through levels of immunity in adult animals and the age range during which different sections of the farming complex and also the spread at seroconversion occurred was defined. Using this information, two associated farms that had only growing pigs. Epidemiological a management program was developed that led to the successful studies demonstrated that MenV spread through the population eradication of MenV from the piggery. This involved cleaning and relatively slowly and that young pigs became infected from about disinfection of pig sheds, and then re-populating with pigs of a 12–16 weeks of age but were consistently immune by the time that single age, usually from the time of weaning when they still had high they had reached breeding age, resulting in a natural cessation of maternal antibody titres that were expected to provide additional the disease. The patterns and rate of spread of MenV infection protection from infection. To eliminate in utero infection, before among pigs suggested that transmission was more likely to be becoming pregnant, sows known to be immune were placed in oro-nasal from faecal material or urine rather than via aerosols sheds that had been cleaned and disinfected. Not only did their from the respiratory tract. immunity prevent new infections but their progeny also had passively acquired immunity. Collectively these measures provided Public health and anxious weeks ahead additional time for any residual virus to become inactivated in the environment and also a long time interval before young pigs had As a part of the epidemiological studies, serum samples were lost protective antibody and become susceptible to infection. collected from all workers on the affected farms, regular visitors, Successful eradication was confirmed by demonstrating that sub- including medical researchers involved in xenotransplantation sequent generations of pigs remained seronegative after losing studies and abattoir employees. Two seropositive male farm work- maternally derived antibodies. ers were identified6. Follow-up medical examinations revealed that both had experienced a severe febrile illness lasting for 10–14 days, with severe headaches and myalgia. Although one person lost a Where is Menangle virus today? considerable amount of body weight, both eventually returned to To the best of our knowledge, there are no terrestrial animals normal health. During sampling of staff, a young female worker currently infected with Menangle virus, but it is still circulating in confided that she had recently become pregnant but had not the fruit bat population. The emergence of other zoonotic viruses advised anyone. Although seronegative, she remained on stress- such as the Hendra-related Nipah virus from Pteropus spp in SE related leave until after the birth of a normal child. Asia, has provided additional incentive to minimise opportunities for contact of terrestrial animals with environments contaminated Origin of the virus by excreta from flying foxes. Had there not been rapid control of spread through the implementation of farm quarantine and The close proximity of a large colony of grey-headed flying foxes control measures, MenV could have spread throughout the (Pteropus poliocephalus) to the piggery and the recent discovery Australian pig population and, with amplification of the virus in of Hendra virus in Australia raised suspicion that flying foxes could pigs, posed an ongoing human occupational health hazard. be a source of MenV. Serological evidence of MenV infection was detected in samples from four different species of flying fox at various locations in Australia, including samples taken from two References et al P. poliocephalus colonies in the vicinity of the affected piggery3,5. 1. Love, R.J. . (2001) Reproductive disease and congenital malformations caused by Menangle virus in pigs. Aust. Vet. J. 79,192–198. doi:10.1111/j.1751- More recently, MenV has been isolated from the urine of flying 0813.2001.tb14578.x

MICROBIOLOGY AUSTRALIA * MARCH 2017 23 Under the Microscope

2. Philbey, A.W. et al. (2007) Skeletal and neurological malformations in pigs Biography congenitally infected with Menangle virus. Aust. Vet. J. 85,134–140. doi:10.1111/ j.1751-0813.2007.00131.x Dr Peter Kirkland is the Head of the Virology Laboratory at et al 3. Philbey, A.W. . (1998) An apparently new virus (family Paramyxoviridae) the state government Elizabeth Macarthur Agriculture Institute at infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4,269–271. doi:10.3201/eid0402.980214 Menangle NSW. Dr Kirkland has had a long career in diagnostic and 4. Bowden, T.R. et al. (2001) Molecular characterization of Menangle virus, a novel research projects in animal health. He has been instrumental in the paramyxovirus which infects pigs, fruit bats, and humans. Virology 283,358–373. fi doi:10.1006/viro.2001.0893 identi cation of several new viruses, including Menangle virus that 5. Kirkland, P.D. et al. (2001) Epidemiology and control of Menangle virus in pigs. was transmitted from flying foxes to pigs, a novel that was Aust. Vet. J. 79,199–206. doi:10.1111/j.1751-0813.2001.tb14580.x responsible for a major disease outbreak in pigs and viruses that 6. Chant, K. et al. (1998) Probable human infection with a newly described virus in the family Paramyxoviridae. Emerg. Infect. Dis. 4,273–275. doi:10.3201/eid0402. have caused blindness and sudden deaths in macropods. In 2007 he 980215 led the EMAI team during the diagnosis and response to the equine et al 7. Barr, J.A. . (2012) Evidence of bat origin for Menangle virus, a zoonotic fl paramyxovirus first isolated from diseased pigs. J. Gen. Virol. 93, 2590–2594. in uenza outbreak and in 2011 the investigation of the large West doi:10.1099/vir.0.045385-0 Nile virus outbreak in horses in NSW. His research interests include et al 8. Breed, A.C. . (2010) Prevalence of Henipavirus and Rubulavirus antibodies in vector borne viruses and the development and evaluation of rapid Pteropid bats, . Emerg. Infect. Dis. 16, 1997–1999. doi:10.3201/ eid1612.100879 diagnostic assays for viral diseases of animals.

24 MICROBIOLOGY AUSTRALIA * MARCH 2017 Under the Microscope

Virus discovery in bats

Rebecca I Johnson Ina L Smith CSIRO Australian Animal CSIRO Australian Animal Health Laboratory Health Laboratory 5 Portarlington Road 5 Portarlington Road East Geelong, Vic. 3219, Australia East Geelong, Vic. 3219, Australia Email: [email protected] Email: [email protected]

Comprising approximately 20% of known mammalian generation sequencing is not suitable for all experimental aims, 1 species, bats are abundant throughout the world .In such as when the targeted discovery of particular viral families is recent years, bats have been shown to be the reservoir host required19. for many highly pathogenic viruses, leading to increased attempts to identify other zoonotic bat-borne viruses. These The bat sampling method can affect which viruses are able to be efforts have led to the discovery of over 200 viruses in bats detected and can result in a bias towards particular families of and many more viral sequences from 27 dif- viruses. The bat specimen used for discovery is an important ferent viral families2,3 (Table 1). Over half of the world’s consideration, as well as the time of year these specimens are recently emerged infectious diseases originated in wild- collected, the intervals between collections, the species of bat to be life15, with the genetic diversity of viruses greater in bats targeted and the ecology of the bat species, especially as not all than in anyother animal16. Ashumans continue toencroach viruses are continually shed in the population. In the case of on the habitat of bats, the risk of spillover of potentially Marburg virus, peaks of shedding were seen during birthing sea- zoonotic viruses is also continuing to increase. Therefore, sons as these months coincided with a peak in infection in 6-month- 20 the surveillance of bats and discovery of novel pathogens old juvenile bats . is necessary to prepare for these spillover events17. Although lethal sampling of bats may be necessary for virus dis- Not only does virus discovery increase our understanding of the covery from particular viral families, non-lethal sampling has role that bats play in emerging infectious diseases, it also allows the resulted in the discovery of a greater number of novel viruses 18 development of diagnostic tools resulting in a much more efficient across a similar number of studies . Bat urine and faeces have been response if a spillover event occurred, reducing both the economic favoured as non-invasive samples for virus discovery, however and public health impact of the virus. Virus discovery is important active bat catching and sampling can give more accurate calcula- for identifying potential zoonotic threats and can assist with the tions of viral prevalence. In the case of Hendra virus, urine was the characterisation of already emerged zoonotic viruses, as well as most significant form of virus transmission, with higher titres of providing phylogenetic evidence for the origin and evolution of virus seen in urine compared to specimens such as nasal swabs, 21 these viruses; for example the potential bat origin of primate faecal samples and serum . Pooled urine can be collected from hepadnaviruses5. plastic sheets laid below bat colonies and stored in a viral transport medium at À808C for later analysis22. These samples can then Advancements in technology have also contributed to the in- be analysed in multiple different ways depending on the chosen creased rate of virus discovery, with molecular techniques now method for virus discovery. overtaking serological methods and virus isolation18. Improvement in the accessibility of next generation sequencing has allowed the Molecular techniques such as pan-viral family PCR are useful for development of unbiased methods of analysing bat specimens as targeted discovery of viruses. This involves amplifying a region of well as more rapid characterisation of novel viruses. However, next the genome that is highly conserved across that viral family using

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17008 25 Under the Microscope

Table 1. Summary of viral families detected in bats2,4 and their zoonotic pan-viral family PCR detected paramyxovirus sequences, including potential. Viral families were classed as containing zoonoses if any of the viruses detected in bats had been associated with disease in in bats that yielded no positive results when their pooled serum 4–14 humans . samples were analysed by next generation sequencing. However, it Virus family Zoonotic is possible that the negative results by next generation sequencing ssRNA (negative Arenaviridae – were due to low concentrations of virus in the blood rather than sense) significantly lower sensitivity25. The primers utilised by pan-viral – family PCR can only detect viruses that are related to previously + Bunyaviridae identified viruses. In an attempt to reduce the bias introduced by Filoviridae + sequence specific primers, multiple different primer sets and methods can be utilised for the same samples25. Although this Orthomyxoviridae + approach has led to the discovery of many novel viruses, other Paramyxoviridae + methods provide a hypothesis-free approach. + Next generation sequencing has become increasingly more acces- ssRNA (positive – Astroviridae sible as a method for virus discovery, although it is still more sense) – expensive than other molecular methods and requires bioinfor- matics knowledge to correctly analyse the raw data and generate a Coronaviridae + consensus genome19. When correctly designed, metagenomic – analysis of bat specimens can allow the hypothesis free discovery fi – of many novel viruses, including those that are signi cantly diver- gent from previously identified viruses. The high throughput Flaviviridae + technique also allows efficient screening of a large number of bat – specimens. This method was used to identify highly divergent novel

Picornaviridae – in bats in Cameroon that were unlikely to have been successfully detected using the currently available primer combi- Togaviridae + nations26. The sensitivity of high throughput sequencing is con- dsDNA Adenoviridae – tinuing to improve for virus discovery, employing techniques such as positive enrichment of samples for virus sequences using – probes that cover the genomes of all the viral taxa known to infect – vertebrates27. However, this enrichment may reduce the likelihood Herpesviridae – of discovering novel viruses.

– Virus isolation, supported by other molecular detection techni- – ques, continues to play a significant role in the discovery of novel viruses as it allows further characterisation and comparison with – other viruses. Virus isolation followed by pan-family PCR was – Poxviridae successfully used for the surveillance of Australian pteropid bats 22 dsRNA Reoviridae + and resulted in the discovery of multiple novel paramyxoviruses . However, not all viruses cause obvious in cell Picobirnaviridae – culture, making it difficult to detect virus growth in cells. Further- – more, the viruses may require very specific cell lines and conditions fl Retro-transcribing + for growth, if they can even be cultured at all. Bat derived in uenza viruses have been detected in Sturnira lilium in Guatemala by pan- Retroviridae – influenza virus RT-PCR28, but subsequent attempts at culturing were challenging, due in part to their divergent surface proteins degenerate primers23,24. In one study, this approach was employed and unique basolateral cell entry mechanism29,30. In vivo isolation to detect sequences of 66 new viruses from the Paramyxoviridae methods may also be used, such as the use of suckling mice or family from bats and rodents around the world25. In this example, knockout mice31.

26 MICROBIOLOGY AUSTRALIA * MARCH 2017 Under the Microscope

Virus discovery from bats increases our database of known viruses 16. Li, Y. et al. (2010) Host range, prevalence, and genetic diversity of adenoviruses J. Virol. 84 – and is necessary for preparing a rapid response to emerging in bats. , 3889 3897. doi:10.1128/JVI.02497-09 et al – 17 17. Daszak, P. . (2000) Emerging infectious diseases of wildlife threats to infectious diseases . For example, the isolation and characterisa- biodiversity and human health. Science 287, 443–449. doi:10.1126/science. tion of Hendra virus in 1994 enabled the development of diagnostic 287.5452.443 PLoSOne 11 assays that played an important role in the identification of Nipah 18. Young, C.C. and Olival, K.J.(2016) Optimizing viral discovery in bats. , e0149237. doi:10.1371/journal.pone.0149237 virus during an outbreak of encephalitic disease five years later. 19. Radford, A.D. et al. (2012) Application of next-generation sequencing technol- Cross-reactivity with antibodies to Hendra virus was observed ogies in virology. J. Gen. Virol. 93,1853–1868. doi:10.1099/vir.0.043182-0 during initial screening against the unknown virus causing fatal 20. Amman, B.R. et al. (2012) Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human disease in pigs and humans. Then, primers developed against infection. PLoS Pathog. 8, e1002877. doi:10.1371/journal.ppat.1002877 32 Hendra virus assisted in determining the sequence of Nipah virus . 21. Edson, D. et al. (2015) Routes of Hendra virus excretion in naturally-infected fl PLoS One 10 Virus discovery can also facilitate the development of diagnostic ying-foxes: implications for viral transmission and spillover risk. , e0140670. doi:10.1371/journal.pone.0140670 tools and further research into pathogenic determinants of other 22. Barr, J. et al. (2015) Isolation of multiple novel paramyxoviruses from pteropid bat viruses. It is estimated that each bat species would have to be urine. J. Gen. Virol. 96,24–29. doi:10.1099/vir.0.068106-0 et al sampled 7000 times before the viral diversity limit is reached33,so 23. Tong, S. . (2008) Sensitive and broadly reactive reverse transcription-PCR assays to detect novel paramyxoviruses. J. Clin. Microbiol. 46, 2652–2658. with approximately 1200 species of bat around the world, the doi:10.1128/JCM.00192-08 discovery of novel viruses in bats has a long way to go. 24. Rose, T.M. (2005) CODEHOP-mediated PCR – a powerful technique for the identification and characterization of viral genomes. Virol. J. 2, 20. doi:10.1186/ 1743-422X-2-20 References 25. Drexler, J.F. et al. (2012) Bats host major mammalian paramyxoviruses. Nat. 1. Churchill, S. (1998) Australian Bats. Reed New Holland, Sydney, Australia. Commun. 3, 796. doi:10.1038/ncomms1796 2. Moratelli, R. and Calisher, C.H. (2015) Bats and zoonotic viruses: can we 26. Yinda, C.K. et al. (2016) Novel highly divergent reassortant bat rotaviruses in confidently link bats with emerging deadly viruses? Mem. Inst. Oswaldo Cruz Cameroon, without evidence of . Sci. Rep. 6, 34209. doi:10.1038/ 110,1–22. doi:10.1590/0074-02760150048 srep34209 3. Chen, L. et al. (2014) DBatVir: the database of bat-associated viruses. Database: 27. Briese, T. et al. (2015) Virome capture sequencing enables sensitive viral diag- the journal of biological databases and curation 2014, bau021. doi:10.1093/ nosis and comprehensive virome analysis. MBio 6, e01491–15. doi:10.1128/ database/bau021 mBio.01491-15 4. Luis, A.D. et al. (2013) A comparison of bats and rodents as reservoirs of zoonotic 28. Tong, S. et al. (2012) A distinct lineage of influenza A virus from bats. Proc. Natl. viruses: are bats special? Proc. Biol. Sci. 280, 20122753. doi:10.1098/rspb.2012. Acad. Sci. USA 109, 4269–4274. doi:10.1073/pnas.1116200109 2753 29. Sun, X. et al. (2013) Bat-derived influenza H17 does not bind 5. Drexler, J.F. et al. (2013) Bats carry pathogenic hepadnaviruses antigenically canonical avian or human receptors and most likely uses a unique entry mech- related to hepatitis and capable of infecting human hepatocytes. Proc. anism. Cell Reports 3,769–778. doi:10.1016/j.celrep.2013.01.025 Natl. Acad. Sci. USA 110 – , 16151 16156. doi:10.1073/pnas.1308049110 30. Moreira, E.A. et al. (2016) Synthetically derived bat influenza A-like viruses reveal 6. Dacheux, L. et al. (2014) A preliminary study of viral of French a cell type- but not species-specific tropism. Proceedings of the National Academy bat species in contact with humans: identification of new mammalian viruses. of Sciences of the United States of America. PLoS One 9 , e87194. doi:10.1371/journal.pone.0087194 31. Lipkin, W.I. (2010) Microbe hunting. Microbiology and molecular biology reviews 7. Cogswell-Hawkinson, A. et al. (2012) Tacaribe virus causes fatal infection of Microbiol. Mol. Biol. Rev. 74, 363–377. doi:10.1128/MMBR.00007-10 J. Virol. 86 – an ostensible reservoir host, the Jamaican fruit bat. , 5791 5799. 32. Harcourt, B.H. et al. (2000) Molecular characterization of Nipah virus, a newly doi:10.1128/JVI.00201-12 emergent paramyxovirus. Virology 271,334–349. doi:10.1006/viro.2000.0340 et al 8. Tse, H. . (2012) Discovery and genomic characterization of a novel bat 33. Anthony, S.J. et al. (2013) A strategy to estimate unknown viral diversity in PLoS One with unusual genomic features and phylogenetic position. mammals. MBio 4, e00598–13. doi:10.1128/mBio.00598-13 7, e34987. doi:10.1371/journal.pone.0034987 9. Drexler, J.F. et al. (2012) Bats worldwide carry virus-related viruses that form a putative novel genus within the family Hepeviridae. J. Virol. 86, 9134–9147. doi:10.1128/JVI.00800-12 10. Baker, K.S. and Murcia, P.R. (2014) Poxviruses in bats ... so what? Viruses 6, Biographies 1564–1577. doi:10.3390/v6041564 Rebecca I Johnson is a PhD student at the CSIRO Australian 11. Lima, F.E. et al. (2015) Genomic characterization of novel circular ssDNA viruses from insectivorous bats in Southern Brazil. PLoS One 10, e0118070. doi:10.1371/ Animal Health Laboratory. She has an interest in emerging diseases journal.pone.0118070 and her work focuses on the characterisation of novel viruses et al 12. Canuti, M. . (2011) Two novel parvoviruses in frugivorous New and Old World isolated from pteropid bats. bats. PLoS One 6, e29140. doi:10.1371/journal.pone.0029140 13. Cibulski, S.P. et al. (2014) A novel anelloviridae species detected in Tadarida Ina L Smith is a senior research scientist at the CSIRO Australian brasiliensis bats: first sequence of a Chiropteran Anellovirus. Genome Announc. 2, e01028-14. doi:10.1128/genomeA.01028-14 Animal Health Laboratory. With a strong background in traditional 14. Yang, X. et al. (2012) A novel -like virus isolated from bat . Arch. and molecular virology, her research has focused on viral discovery Virol. 157, 1093–1099. doi:10.1007/s00705-012-1278-y and characterisation, primarily from bats but also from mammals 15. Jones, K.E. et al. (2008) Global trends in emerging infectious diseases. Nature 451, 990–993. doi:10.1038/nature06536 and .

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Bats, bacteria and their role in health and disease

Europe3. They were classified as members of the genus Vesperti- liibacter and provide some evidence for species-specific adapta- tions in bats. Three further isolates that also belong to this genus Kristin Mühldorfer have been collected recently from Nathusius’s pipistrelles (Pipis- Leibniz Institute for Zoo and Wildlife trellus nathusii) confirming the presence of diverse bacterial Research strains in different species of insectivorous bats (family Vesperti- Department of Wildlife Diseases Alfred-Kowalke-Str. 17, 10315 lionidae) (Figure 1). Berlin, Germany Tel: +49 30 5168-215 Bartonella, Leptospira and many Pasteurellaceae species are Email: [email protected] known pathogens that can cause serious bacterial infections in mammals. Infected bats, however, appear to be healthy or do not show specific disease symptoms suggesting that these bacteria 4 Bats are ancient and among the most diverse mammals in might possibly have co-evolved with their hosts . To date, bacterial 5 terms of species richness, diet and habitat preferences, diseases are rarely described in wild bats . They often represent characteristics that may contribute to a high diversity of individual cases that have been caused by well-recognised bacterial infectious agents. During the past two decades, the interest pathogens such as Pasteurella multocida, Escherichia coli, in bats and their microorganisms largely increased because Salmonella and Yersinia species. The only exception known so of their role as reservoir hosts or carriers of important far is an outbreak of acute systemic pasteurellosis caused by pathogens. Rapid advances in microbial detection and char- P. multocida in a colony of wild big brown bats (Eptesicus fuscus) 6 acterisation by high-throughput sequencing technologies in Wisconsin, USA, which resulted in high mortality . In fruit bats have led to large genetic data sets but also improved our kept in captivity sporadic infections associated with different possibilities and speed of identifying unknown infectious opportunistic bacteria (e.g. Staphylococcus aureus, Listeria or agents. Assessing the risk of infectious diseases in bats and alpha-hemolytic Streptococcus spp.) (Lisa L Farina, pers. comm.) 7 their pathological manifestation, however, is still challeng- and serious outbreaks of Yersinia pseudotuberculosis are known ing because of limited access to appropriate material and to occur. field data, and continuing limitations in wildlife diagnostics and the interpretation of genetic results. As a consequence, 93-5-16 Pipistrellus nathusii emerging pathogens can suddenly appear with devastating 92 effects as happened for the white nose syndrome. To date, E157-08 Pipistrellus pipistrellus 2% much research on bats and infectious agents still focusses 78 E127-08 Nyctalus noctula on viruses, whilst the knowledge on bacteria and their role 88 E40-12 Nyctalus noctula in disease is comparatively low. 95

E146-08 Nyctalus noctula Some bacterial pathogens, such as Bartonella and Leptospira, have 100 been more intensively studied and it seems that bats have a 92-1-16 Pipistrellus nathusii 98 remarkably high diversity of bacterial species1,2. Interestingly, 93-1-16 Pipistrellus nathusii genetic analyses provide continuous evidence of yet-undescribed 97 bacterial sequences and clades that might be associated with E145-08 Eptesicus serotinus different species of bats, their habitats, ectoparasites (only for 322-4-16 Epomophorus gambianus Bartonella) or geographic origins. It is very likely that they repre- Figure 1. based on partial 16S rRNA gene sequences. sent novel species within the families Bartonellaceae and Leptos- Sequences of Vespertiliibacter strains isolated from wild insectivorous bats of five different vespertilionid species were compared to a piraceae but need to be further characterised. sequence of an unknown Pasteurellaceae-like bacterium isolated from a Gambian epauletted fruit bat kept in a zoo. The tree was built Novel bacteria belonging to the family of Pasteurellaceae were in Bionumerics v. 7.0 (Applied Maths) using the UPGMA method. Bootstrap values of 500 replicates are indicated at the branch points. isolated from three different species of vespertilionid bats from The bar represents 2% sequence divergence.

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The limited number of studies and literature available on infectious 3. Mühldorfer, K. et al. (2014) Proposal of Vespertiliibacter pulmonis gen. nov., sp. diseases of bats most probably reflect challenges we face in wildlife nov. and two genomospecies as new members of the family Pasteurellaceae isolated from European bats. Int. J. Syst. Evol. Microbiol. 64, 2424–2430. health investigations. For example, bats roosting and foraging in doi:10.1099/ijs.0.062786-0 close proximity to humans are more likely to be found than bats that 4. Lei, B.R. and Olival, K.J. (2014) Contrasting patterns in mammal-bacteria coevo- lution: Bartonella and Leptospira in bats and rodents. PLoS Negl. Trop. Dis. 8, inhabit remote areas. Predation, scavenging and the fast decom- e2738. doi:10.1371/journal.pntd.0002738 position of bat carcasses limit the access to diseased or dead animals 5. Mühldorfer, K. (2013) Bats and bacterial pathogens: a review. Zoonoses Public Health 60 – and strongly affect wildlife diagnostics. High-throughput sequenc- ,93 103. doi:10.1111/j.1863-2378.2012.01536.x 6. Blehert, D.S. et al. (2014) Acute pasteurellosis in wild big brown bats (Eptesicus ing has markedly enhanced our abilities to detect unknown, non- fuscus). J. Wildl. Dis. 50,136–139. doi:10.7589/2012-02-063 culturable infectious agents or to study host-associated microbial 7. Nakamura, S. et al. (2013) Outbreak of yersiniosis in Egyptian rousette bats communities in bats8. The genetic analyses are complex and (Rousettus aegyptiacus) caused by Yersinia pseudotuberculosis serotype 4b. J. Comp. Pathol. 148, 410–413. doi:10.1016/j.jcpa.2012.07.007 generate large amounts of sequencing data that represent numer- 8. Phillips, C.D. et al. (2012) Microbiome analysis among bats describes influences ous bacterial taxa (host microbiota, unknown bacterial species, of host phylogeny, life history, physiology and geography. Mol. Ecol. 21, 2617– ingested and environmental bacteria) and require judicious inter- 2627. doi:10.1111/j.1365-294X.2012.05568.x 9. Dietrich, M. et al. (2017) The excreted microbiota of bats: evidence of niche pretation of results. Clearly, they provide valuable insights into the specialisation based on multiple body habitats. FEMS Microbiol. Lett. 364, composition of the microflora and the bacterial diversity in differ- doi:10.1093/femsle/fnw284 fl ent species of bats9,10. In perspective, basic knowledge about bats 10. Avena, C.V. (2016) Deconstructing the bat skin microbiome: in uences of the host and the environment. Front. Microbiol. 7, 1753. doi:10.3389/fmicb.2016. and their microorganisms will enhance our understanding of 01753 existing bacteria-host interactions and their role in health and disease. Biography Acknowledgement Kristin Mühldorfer is a scientist in the Department of Wildlife Diseases at the Leibniz Institute for Zoo and Wildlife Research in Thanks to Nadine Jahn for her technical assistance. Berlin, Germany. She is a certified veterinary microbiologist and References head of the bacteriology laboratory. She conducts research and fi 1. McKee, C.D. et al. (2016) Phylogenetic and geographic patterns of Bartonella teaches in the eld of wildlife microbiology and infectious diseases. host shifts among bat species. Infect. Genet. Evol. 44, 382–394. doi:10.1016/ Her main interests are in bacteria and fungi of captive and free-living j.meegid.2016.07.033 wildlife species from disease ecology, evolutionary adaptation and 2. Dietrich, M. et al. (2015) Leptospira and bats: story of an emerging friendship. PLoS Pathog. 11, e1005176. doi:10.1371/journal.ppat.1005176 conservation points of view.

MICROBIOLOGY AUSTRALIA * MARCH 2017 29 Under the Microscope

The interplay between viruses and the immune system of bats

Michelle L Baker Stacey Leech CSIRO, Australian Animal Health Laboratory CSIRO, Australian Animal Geelong, Vic. 3220, Australia Health Laboratory Tel: +61 3 5227 5052 Geelong, Vic. 3220, Australia Email: [email protected]

Bats are an abundant and diverse group of mammals with The availability of bat genome and transcriptome datasets from an array of unique characteristics, including their well- a number of bat species has accelerated the identification of key known roles as natural reservoirs for a variety of viruses. components of the immune system17,19,21–25. These studies have These include the deadly zoonotic paramyxoviruses; also provided evidence for a link between flight and immunity with Hendra (HeV) and Nipah (NiV)1,2, lyssaviruses3, coronaviruses the inadvertent selection on key immune genes and pathways as a such as severe acute respiratory coronavirus (SARS-CoV)4 consequence of the evolution of flight19. Functional studies in vitro and filoviruses such as Marburg5. Although these viruses are have demonstrated that differences in the innate immune system of highly pathogenic in other species, including humans, bats bats play a key role in controlling . In particular, rarely show clinical signs of disease whilst maintaining the components of the interferon (IFN) system, including IFN-alpha ability to transmit virus to susceptible vertebrate hosts. In (IFNa), IFN signalling molecules and IFN stimulated genes are addition, bats are capable of clearing experimental infec- constitutively expressed in unstimulated pteropid bat tissues and tions with henipaviruses, filoviruses and lyssaviruses at cells15,17. Although the IFN system provides the first line of defence doses of infection that are lethal in other mammals6–12. againstinfection, bats are the only species known that constitutively Curiously, the ability of bats to tolerate viral infections does activate their IFN response in the absence of infection, providing not appear to extend to extracellular pathogens such as evidence that the baseline activation of the innate immune system bacteria, fungi and parasites13. Over the past few years, of bats is considerably higher than that of other mammals. Pre- considerable headway has been made into elucidating the sumably, the constitutive expression of IFN allows bats to avoid the mechanisms responsible for the ability of bats to control lag time between infection and immune activation, thus providing a viral replication, with evidence for unique differences in more immediate response. However, prolonged exposure to IFN is the innate immune responses of bats14–20. However, many generally associated with pathological side effects in other spe- questions remain around mechanisms responsible for the cies26. Elucidating how bats maintain IFNa expression in the ability of bats to co-exist with viruses, including their ability absence of inflammation could therefore have important implica- to tolerate constitutive immune activation, the triggers tions for treating viral infections in humans and other species. associated with viral spillover events and the sites of viral Clearly, much remains to be learned about the unique innate replication. Although bats appear to have all of the major immune response of bats. components of the immune system present in other species, their unique ecological characteristics (including flight, Much of what we know about the kinetics of the immune response high density populations and migration) combined with of bats to viruses in vivo has been obtained from experimental their long co-evolutionary history with viruses has likely infections. With the exception of rabies and lyssavirus infections, shaped their immune response resulting in an equilibrium these experiments have demonstrated that bats rapidly clear in- – between the host and its pathogens. fection with no clinical disease6 9,11,12,27,28. Experimental infections

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wild caught bats and spillover of viruses from bats to other sus- ceptible species on an ongoing basis3,4,36–38. Thus, viruses appear to be constantly circulating in bat populations at some level, either as a consequence of ongoing infections of naïve individuals, through oscillating or though episodic shedding from persistently infected individuals39. Natural cycles driven by environmental (e.g. climate, food availability, human activity) and internal triggers (e.g. mating, birthing, lactation) have been hypothesised to dampen the bat’s immune response, leading to increased viral replication and consequently to increased spillover – events39 41. However, evidence to link specific physiological or ecological triggers with changes in immune responses and viral Figure 1. Captive Pteropus alecto. (Photo courtesy of Susanne Wilson, replication is currently lacking and considerable work is still re- CSIRO.) quired to fully understand the relationship between viruses and the ability of the immune response of bats to control replication. of bats with the henipaviruses; HeV and NiV have provided the most This information will lead to new insights for the prediction and information to date regarding the kinetics of infection. Pteropid prevention of spillover events and for the treatment of infectious bats (Figure 1) infected with henipaviruses have detectible viral diseases in humans and other species. RNA in rectal and throat swabs from 2–7 days post infection (dpi) and in urine and blood samples collected after 7 dpi8. However, viremia is typically short and viral antigen is generally undetectable in tissues by 21 dpi8,11. Although antibody responses have been the References only immune parameter measured (due partly to the lack of bat 1. Eaton, B.T. et al. (2006) Hendra and Nipah viruses: different and dangerous. specific reagents) these studies have provided important evidence Nat. Rev. Microbiol. 4,23–35. doi:10.1038/nrmicro1323 Bats and for the ability of bats to rapidly clear viral infections. Virus specific 2. Anderson, D.E. and Marsh, G.A. (2015) Bat paramyxoviruses. In Viruses, pp. 99–126, John Wiley & Sons, Inc. antibodies have been detected in wild caught bats indicating 3. Kuzmin, I.V. and Rupprecht, C.E. (2015) Bat lyssaviruses. In Bats and Viruses, – that they are capable of generating an antibody response29 33. pp. 47–97, John Wiley & Sons, Inc. et al Bats and Viruses – However, evidence from experimental infections have shown 4. Ge, X.-Y. . (2015) Bat coronaviruses. In , pp. 127 155, John Wiley & Sons, Inc. the development of neutralising antibodies is often delayed and 5. Maganga, G.D. et al. (2015) Bat filoviruses. In Bats and Viruses, pp. 157–175, some animals fail to generate an antibody response within the John Wiley & Sons, Inc. timeframe of the experiment8,11,12,27,28. This coupled with the 6. Jones, M.E. et al. (2015) Experimental inoculation of Egyptian rousette bats (Rousettus aegyptiacus) with viruses of the Ebolavirus and Marburgvirus constitutive activation of components of the innate immune system genera. Viruses 7, 3420–3442. doi:10.3390/v7072779 may mean that the adaptive immune response in bats is less critical. 7. Middleton, D.J. et al. (2007) Experimental Nipah virus infection in Pteropid bats Pteropus poliocephalus J. Comp. Pathol. 136 – Much less is known about T cell mediated immunity in bats and no ( ). , 266 272. doi:10.1016/j.jcpa. 2007.03.002 studies have examined T cell responses in experimentally infected 8. Halpin, K. et al. (2011) Pteropid bats are confirmed as the reservoir hosts of bats. However, differences in the repertoire of major histocom- Henipaviruses: a comprehensive experimental study of virus transmission. Am. J. Trop. Med. Hyg. 85, 946–951. doi:10.4269/ajtmh.2011.10-0567 patibility complex (MHC) class I molecules and the self and 9. Amman, B.R. et al. (2015) Oral shedding of Marburg virus in experimentally HeV they present already point to differences in T cell infected Egyptian fruit bats (Rousettus aegyptiacus) J. Wildl. Dis. 51, 113–124. mediated responses to infection34,35. Thus, like the innate immune doi:10.7589/2014-08-198 10. Sétien, A.A. et al. (1998) Experimental rabies infection and oral vaccination response, the cell mediated response may also have undergone in vampire bats (Desmodus rotundus). Vaccine 16, 1122–1126. doi:10.1016/ changes associated with the long co-evolutionary history of bats S0264-410X(98)80108-4 with viruses. 11. Williamson, M.M. et al. (1998) Transmission studies of Hendra virus (equine morbilli-virus) in fruit bats, horses and cats. Aust. Vet. J. 76, 813–818. doi:10.1111/ j.1751-0813.1998.tb12335.x The fine balance between the immune system of bats and their 12. Williamson, M.M. et al. (2000) Experimental Hendra virus infection in pregnant viruses has presumably achieved an equilibrium that is able to guinea-pigs and fruit bats (Pteropus poliocephalus). J. Comp. Pathol. 122, support both the survival of the host and low level replication of the 201–207. doi:10.1053/jcpa.1999.0364 ‘ ’ pathogen. Despite the seemingly high activation of the bat’s innate 13. Brook, C.E. and Dobson, A.P. (2015) Bats as special reservoirs for emerging zoonotic pathogens. Trends Microbiol. 23, 172–180. doi:10.1016/j.tim.2014.12. immune system, viruses have been isolated from naturally infected 004

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14. Zhou, P. et al. (2013) Bat Mx1 and Oas1, but not Pkr are highly induced by bat 31. Leroy, E.M. et al. (2005) Fruit bats as reservoirs of Ebola virus. Nature 438, interferon and viral infection. Dev. Comp. Immunol. 40, 240–247. doi:10.1016/ 575–576. doi:10.1038/438575a j.dci.2013.03.006 32. Machain-Williams, C. et al. (2013) Serologic evidence of flavivirus infection in 15. Zhou, P. et al. (2014) IRF7 in the Australian black flying fox, Pteropus alecto: bats in the Yucatan Peninsula of Mexico. J. Wildl. Dis. 49, 684–689. doi:10.7589/ evidence for a unique expression pattern and functional conservation. PLoS One 2012-12-318 9 , e103875. doi:10.1371/journal.pone.0103875 33. Peel, A.J. et al. (2012) Henipavirus neutralising antibodies in an isolated island 16. He, X. et al. (2014) Anti-lyssaviral activity of interferon k and w from the Serotine population of African fruit bats. PLoS One 7, e30346. doi:10.1371/journal.pone. bat, Eptesicus serotinus. J. Virol. 88, 5444–5454. doi:10.1128/JVI.03403-13 0030346 17. Zhou, P. et al. (2016) Contraction of the type I IFN locus and unusual constitutive 34. Ng, J.H.J. et al. (2016) Evolution and comparative analysis of the bat MHC-I region. expression of IFN-a in bats. Proc. Natl. Acad. Sci. USA 113, 2696–2701. Sci. Rep. 6, 21256. doi:10.1038/srep21256 doi:10.1073/pnas.1518240113 35. Wynne, J.W. et al. (2016) Characterization of the antigen processing machinery 18. Zhou, P. et al. (2011) Type III IFN receptor expression and functional charac- and endogenous peptide presentation of a bat MHC class I molecule. J. Immunol. terisation in the Pteropid bat, Pteropus alecto. PLoS One 6, e25385. doi:10.1371/ 196, 4468–4476. doi:10.4049/jimmunol.1502062 journal.pone.0025385 36. Clayton, B.A. et al. (2013) Henipaviruses: an updated review focusing on the 19. Zhang, G. et al. (2013) Comparative analysis of bat genomes provides insight Pteropid reservoir and features of transmission. Zoonoses Public Health 60, into the evolution of flight and immunity. Science 339, 456–460. doi:10.1126/ 69–83. doi:10.1111/j.1863-2378.2012.01501.x science.1230835 37. Luby, S.P. et al. (2009) Recurrent zoonotic transmission of Nipah virus into 20. Cowled, C. et al. (2011) Molecular characterisation of Toll-like receptors in the humans, Bangladesh, 2001–2007. Emerg. Infect. Dis. J. 15, 1229. fl Pteropus alecto Dev. Comp. Immunol. 35 – black ying fox . ,7 18. doi:10.1016/ 38. Amman, B.R. et al. (2012) Seasonal pulses of Marburg virus circulation in juvenile j.dci.2010.07.006 Rousettus aegyptiacus bats coincide with periods of increased risk of human 21. Papenfuss, A.T. et al. (2012) The immune gene repertoire of an important viral infection. PLoS Pathog. 8, e1002877. doi:10.1371/journal.ppat.1002877 fl BMC Genomics 13 reservoir, the Australian black ying fox. , 261. doi:10.1186/ 39. Plowright, R.K. et al. (2016) Transmission or within-host dynamics driving 1471-2164-13-261 pulses of zoonotic viruses in reservoir–host populations. PLoS Negl. Trop. Dis. 22. Shaw, T.I. et al. (2012) Transcriptome sequencing and annotation for the 10, e0004796. doi:10.1371/journal.pntd.0004796 Artibeus jamaicensi PLoS One 7 Jamaican fruit bat ( ). , e48472. doi:10.1371/ 40. Plowright, R.K. et al. (2008) Reproduction and nutritional stress are risk factors journal.pone.0048472 for Hendra virus infection in little red flying foxes (Pteropus scapulatus). Proc. 23. Glennon, N.B. et al. (2015) Transcriptome profiling of the virus-induced innate Biol. Sci. 275, 861–869. doi:10.1098/rspb.2007.1260 Pteropus vampyrus immune response in and its attenuation by Nipah virus 41. Baker, K.S. et al. (2014) Viral antibody dynamics in a chiropteran host. J. Anim. J. Virol. 89 – interferon antagonist functions. , 7550 7566. doi:10.1128/JVI.00302-15 Ecol. 83, 415–428. doi:10.1111/1365-2656.12153 24. Wynne, J.W. et al. (2014) Proteomics informed by transcriptomics reveals Hendra virus sensitizes bat cells to TRAIL mediated apoptosis. Genome Biol. 15, 532. 25. Wynne, J.W. and Tachedjian, M. (2015) Bat Genomics. In Bats and Viruses, Biographies pp. 315-326, John Wiley & Sons, Inc. Dr Michelle Baker is a Research Scientist at CSIRO’s Australian 26. Hertzog, P.J. and Williams, B.R. (2013) Fine tuning type I interferon responses. Cytokine Growth Factor Rev. 24, 217–225. doi:10.1016/j.cytogfr.2013.04.002 Animal Health Laboratory. Her research interests are in the area of 27. Turmelle, A.S. et al. (2010) Host immunity to repeated rabies virus infection in big host-pathogen interactions to emerging infectious diseases, with a brown bats. J. Gen. Virol. 91, 2360–2366. doi:10.1099/vir.0.020073-0 focus on antiviral immunity in bats. 28. McColl, K.A. et al. (2002) Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus). Aust. Vet. J. 80, 636–641. Dr Stacey Leech is a post-doctoral scientist within the compar- doi:10.1111/j.1751-0813.2002.tb10973.x 29. Li, W. et al. (2005) Bats are natural reservoirs of SARS-like coronaviruses. Science ative immunology team working at CSIRO Geelong where her 310, 676–679. doi:10.1126/science.1118391 current research utilises genome engineering tools to investigate et al 30. Halpin, K. . (2000) Isolation of Hendra virus from pteropid bats: a natural the innate immunity of the Australian black flying fox (Pteropus reservoir of Hendra virus. J. Gen. Virol. 81, 1927–1932. doi:10.1099/0022- 1317-81-8-1927 alecto).

32 MICROBIOLOGY AUSTRALIA * MARCH 2017 Under the Microscope

Bat and virus ecology in a dynamic world

David TS Hayman David A Wilkinson Molecular Epidemiology and Public Molecular Epidemiology and Public Health Laboratory (mEpiLab) Health Laboratory (mEpiLab) Hopkirk Research Institute Hopkirk Research Institute Massey University Massey University Private Bag 11-222 Private Bag 11-222 Palmerston North, New Zealand Palmerston North, New Zealand Email: [email protected]

The emergence of infectious diseases caused by bat-associ- processes, we must understand reservoir community structures, ated viruses has had a devastating and wide-reaching effect the spatial and temporal nature of reservoir infection, reservoir on human populations. These viruses include lyssaviruses host ranges and dispersal, as well as other factors that contribute to such as rabies virus, the filoviruses, Ebola (EBOV) and Mar- the ‘force of infection’ received by domestic animal or human burg virus, Severe Acute Respiratory Syndrome (SARS) co- populations10. Rich field-data is essential to fully understand and ronavirus, and the paramyxoviruses, Hendra virus (HeV) thus mitigate the risk from any bat associated zoonotic disease. 1 and Nipah virus (NiV) . As a result bats have been the focus Furthermore, as we progress through the Anthropocene era, bat of substantial research (Fig. 1) and certain cellular and populations meet new, direct and indirect challenges as a result of physiological traits of bats are hypothesised to lead to human population growth. These include climate change, habitat 2,3 4 ‘special’ bat-virus associations (but see Han et al. ). The loss, increasing competitionfor resourcesandphysical danger from anthropogenic changes in the world we live will influence man-made structures such as roads and wind-turbines11. Anthro- 5 human health , including through their impact on bat ecol- pogenic changes will alter bat population dynamics, infection ogy and the viruses within bat populations. Australian peo- dynamics within bat populations, and the contact rates between ple and livestock have been infected by novel bat viruses, such as HeV, Menangle viruses (MenV) and Australian bat lyssavirus (ABLV), and are at the forefront of both epidemi- Paper type ological and virological research efforts into cross-species Modelling Marburg virus transmission events (spillover): here we put some of those Virology 300 Ebola virus efforts and the potential impacts of anthropogenic changes SARS related-CoV on bat-virus ecology under the microscope.

200 Nipah virus The complex nature of bat virus spillover and disease emergence, Count Hendra virus encompassing environmental, ecological and biological factors, is perhaps best addressed through statistical and mathematical 100 Rabies virus modelling frameworks that can integrate disparate data sources to make inferences regarding infection dynamics and risk6–8. Detailed epidemiological surveillance has helped to establish 0 predictive models for disease transmission9. Recent work using 1925 1950 1975 2000 Year HeV as a model has suggested a delineation of five major contrib- Figure 1. Trends in published studies of bats and viruses. Studies citing uting factors to viral spillover: (1) reservoir host distribution and ‘chiroptera’ [bat], ‘virus’, and ‘infection’, excluding ‘experiment’, ‘dynamic’, and ‘model’ (blue, virology) have rapidly increased this density; (2) pathogen shedding from reservoir hosts; (3) environ- century, yet those including ‘dynamic’ and ‘model’ are few (red, mental stability of the pathogen; (4) recipient host exposure; and modelling). Timings of virological studies first linking bats to notable viral infections are shown. Data were extracted from Web of Science, (5) recipient host susceptibility8. To successfully model these ending June 2016.

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17011 33 Under the Microscope

people, their animals, and bats. Each will affect the five major recipient species. Once better characterised, predictive models of contributing factors to viral spillover8. land-use change may increase our understanding of risk, and allow the development of strategies to mitigate the risk of spillover. One of the most obvious anthropogenic changes is land-use change. With most land-use changes come non-native species, and The impact of land-use change, including introduced species, is it can be contact with non-native species that leads to spillover. relatively well characterised for mammalian species, though not for Two obvious examples in Australia are HeV, which replicates the viruses within them. However, other anthropogenic changes and amplifies within horses12, and MenV, which infected pigs prior that impact mammalian hosts remain poorly characterised, for to people13. Both epidemiological and clinical reports highlight example; anthropogenic climate change may impact other aspects potential issues with emerging disease events. The first reported of viral-host dynamics and therefore spillover risk. Plowright et al. outbreak of HeV in Hendra, Brisbane, led clinicians to include performed a longitudinal study of HeV in little red flying foxes poisoning, bacterial, viral, and other exotic diseases as potential (Pteropus scapulatus)22. Serological data showed that pregnant causes before HeV was isolated12. In the case of MenV, the virus and lactating females had significantly higher risk of infection and was isolated from stillborn piglets with deformities at a piggery in implied that HeV is transmitted horizontally and immunity (in- in 1997. Stillbirth, birth defects, and mummified ferred through serology) wanes rapidly. The highest seropreva- foetuses are not uncommon in production systems, so determining lence, however, was observed when bats were nutritionally when novel infections are the cause requires appropriate micro- stressed. Stress may reduce the capacity of bats to respond to viral biological studies. Such spillover events are rare, but whether infections through innate immune pathways2,23, leading to greater direct human infection is likely, for either HeV or MenV, without viral replication and adaptive immune responses. Martin et al. intermediate hosts is unknown and prevention of HeV emergence suggest that the bioclimatic niche of two species (P. alecto and focuses on stopping HeV infection in horses14. How infections then P. conspicillatus) determines the spatial pattern of spillover of persist in novel host populations to facilitate human spillover, such HeV24, whereas the environmental survival of HeV was limited in as for NiV in pigs15, and how they may then perpetuate in human ‘normal’ environmental conditions25. These findings suggest an- populations, such as NiV16 or EBOV17 in people, is then determined thropogenic changes that increase stress, such as habitat loss and by a number of other virological and ecological processes. climate change, may increase HeV risk in the future.

Land-use change also typically leads to native habitat loss and How generalisable these Australian study findings are is yet to be fragmentation. Both processes may change bat behaviour and determined, but from an international perspective Australian bat- distribution. In doing so, these changes will inevitably affect viral systems have been useful models to help understand bat-viral the infection dynamics of the viruses within those populations. ecology and its interaction with anthropogenic change. The find- Plowright et al. modelled the transmission dynamics of HeV in ings can potentially help reduce the threat of viral emergence in our Australian Pteropus bats (fruit bats or flying foxes)18. They devel- dynamic world, and thus save , provided that lessons are oped an ecological model of the HeV dynamics within the popula- learned from them and that sufficient and appropriate action is tions and then used field and laboratory data to provide values for taken to mitigate the drivers of viral spillover. the parameters. The models included urban habituation with decreased migration of bat sub-populations, an observed change References in Australian fruit bat ecology. Models predicted that decreased bat 1. Hayman, D.T.S. (2016) Bats as viral reservoirs. Ann. Rev. Virol. 3,1–609. movement could lead to a decline in population immunity through doi:10.1146/annurev-virology-110615-042203 2. Brook, C.E. and Dobson, A.P. (2015) Bats as ‘special’ reservoirs for emerging reduced transmission among sub-populations, giving rise to more zoonotic pathogens. Trends Microbiol. 23,172–180. doi:10.1016/j.tim.2014. intense outbreaks after HeV reintroduction. Furthermore, analyses 12.004 et al – of spatially varying Eucalypt vegetation indices and weather events 3. Luis, A.D. . (2015) Network analysis of host virus communities in bats and rodents reveals determinants of cross-species transmission. Ecol. Lett. 18, suggest 50% of landscape-scale bat behaviour is driven by Euca- 1153–1162. doi:10.1111/ele.12491 lypt resources19. These results suggest land-use changes interact 4. Han, B.A. et al. (2016) Global patterns of zoonotic disease in mammals. Trends Parasitol. 32, 565–577. doi:10.1016/j.pt.2016.04.007 with bat behaviour to determine HeV risk. Habitat fragmentation 5. Romanelli, C. et al. (2015) Connecting global priorities: biodiversity and human has been linked to other bat-related diseases, including NiV en- health: a state of knowledge review. World Health Organization/Secretariat of the cephalitis20 and EBOV disease21. In general, land-use changes UN Convention on Biological Diversity. et al fi largely affect reservoir host distribution and density, potentially 6. Restif, O. . (2012) Model-guided eldwork: practical guidelines for multi- disciplinary research on wildlife ecological and epidemiological dynamics. Ecol. pathogen shedding from reservoir hosts and contact with novel Lett. 15, 1083–1094. doi:10.1111/j.1461-0248.2012.01836.x

34 MICROBIOLOGY AUSTRALIA * MARCH 2017 Under the Microscope

7. Wood, J.L. et al. (2012) A framework for the study of zoonotic disease emergence 25. Martin, G. et al. (2015) Hendra virus survival does not explain spillover patterns and its drivers: spillover of bat pathogens as a case study. Philos. Trans. R. Soc. and implicates relatively direct transmission routes from flying foxes to horses. Lond. B Biol. Sci. 367, 2881–2892. J. Gen. Virol. 96, 1229–1237. doi:10.1099/vir.0.000073 8. Plowright, R.K. et al. (2015) Ecological dynamics of emerging bat virus spillover. Proc. R. Soc. Lond. B Biol. Sci. 282, 20142124. 9. Streicker, D.G. et al. (2016) Host–pathogen evolutionary signatures reveal dynamics and future invasions of vampire bat rabies. Proc. Natl. Acad. Sci. USA 201606587. Biographies 10. Lloyd-Smith, J.O. et al. (2009) Epidemic dynamics at the human-animal interface. Dr David A Wilkinson studied Molecular and Cellular Biochem- Science 326, 1362–1367. doi:10.1126/science.1177345 istry at University of Oxford, UK obtaining both MBiochem and 11. O’Shea, T.J. et al. (2016) Multiple mortality events in bats: a global review. Mammal Rev. 46,175–190. doi:10.1111/mam.12064 DPhil degrees. He has subsequently used these skills to study 12. Murray, K. et al. (1995) A novel morbillivirus pneumonia of horses and its zoonotic infectious diseases. He utilises a combination of classical transmission to humans. Emerg. Infect. Dis. 1,31–33. doi:10.3201/eid0101. 950107 microbiology, genetics and state-of-the-art genomics to study the 13. Philbey, A.W. et al. (1998) An apparently new virus (family Paramyxoviridae) evolution and epidemiology of disease-causing microorganisms. Emerg. Infect. Dis. 4 – infectious for pigs, humans, and fruit bats. ,269271. After several years studying endemic and emerging bacteria and doi:10.3201/eid0402.980214 Centre de Recherche et de Veille sur 14. Middleton, D. et al. (2014) Hendra virus vaccine, a one health approach to viruses, including from bats, for protecting horse, human, and environmental health. Emerg. Infect. Dis. 20, les Maladies Émergentes dans l’Océan Indien, France, he was 372–379. doi:10.3201/eid2003.131159 awarded a postdoctoral fellowship at the Molecular Epidemiology et al fi 15. Pulliam, J.R. . (2011) Agricultural intensi cation, priming for persistence m and the emergence of Nipah virus: a lethal bat-borne zoonosis. J. R. Soc. Interface and Public Health Laboratory ( EpiLab), Massey University, New 9,89–101. doi:10.1098/rsif.2011.0223 Zealand. In New Zealand he studies a number of systems including 16. Gurley, E.S. et al. (2007) Person-to-person transmission of Nipah virus in a Campylobacter and Leptospira in domestic and wild animals. Bangladeshi community. Emerg. Infect. Dis. 13, 1031–1037. doi:10.3201/eid1307. 061128 17. WHO Ebola Response Team. et al. (2015) West African Ebola epidemic after Dr David Hayman is an Associate Professor in Veterinary Public one year—slowing but not yet under control. N. Engl. J. Med. 372,584–587. doi:10.1056/NEJMc1414992 Health and co-directs the Molecular Epidemiology and Public 18. Plowright, R.K. et al. (2011) Urban habituation, ecological connectivity and Health Laboratory (mEpiLab) at Massey University, New Zealand. fl Pteropus epidemic dampening: the emergence of Hendra virus from ying foxes ( m spp.). Proc. R. Soc. 278,3703–3712. doi:10.1098/rspb.2011.0522 The EpiLab develops and applies new techniques to inform 19. Giles, J.R. et al. (2016) Models of Eucalypt phenology predict bat population flux. decision making and guide the prevention and control of infectious Ecol. Evol. 6 – ,7230 7245. doi:10.1002/ece3.2382 disease and forms part of an OIE collaborating Centre for Veterinary 20. Hahn, M.B. et al. (2014) The role of landscape composition and configuration on Pteropus giganteus roosting ecology and Nipah virus spillover risk in Bangladesh. Epidemiology and Public Health. Previous positions include a Am. J. Trop. Med. Hyg. 90,247–255. doi:10.4269/ajtmh.13-0256 David H Smith Conservation Postdoctoral Fellowship at Colorado et al 21. Rulli, M.C. . (2017) Is there a nexus between deforestation in Africa and Ebola State University and University of Florida, USA; a Wellcome virus disease outbreaks? Sci. Rep. in press. Trust Research Training Fellowship and Cambridge Infectious 22. Plowright, R.K. et al. (2008) Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus). Proc. Diseases Consortium Fellowship at University of Cambridge, UK, Biol. Sci. 275,861–869. from where he obtained PhD. He has worked in the Wildlife 23. Zhou, P. et al. (2016) Contraction of the type I IFN locus and unusual constitutive expression of IFN-a in bats. Proc. Natl. Acad. Sci. USA 113,2696–2701. Zoonoses and Vector-borne Diseases Group at the Animal doi:10.1073/pnas.1518240113 and Plant Health Agency and Institute of Zoology, and holds a et al fl fi 24. Martin, G.A. . (2016) Climatic suitability in uences species speci c abun- veterinary degree from Edinburgh and a Conservation Biology dance patterns of Australian flying foxes and risk of Hendra virus spillover. One Health 2,115–121. doi:10.1016/j.onehlt.2016.07.004 MSc from Kent, UK.

MICROBIOLOGY AUSTRALIA * MARCH 2017 35 ASM Affairs

Bi-State Conference 2016: event report

Edward Fox, Christine Seers and Karena Waller

The 2016 Bi-State Conference was held on 25–26 November in the closed the second session with insights into some recent outbreaks scenic Tasmanian city of Launceston, located at the head of the of bacterial illness linked with dairy foods, notably recent outbreaks picturesque Tamar Valley. The conference venue was Peppers caused by Listeria monocytogenes and Shiga-toxigenic Escherichia Seaport Hotel, situated on the bank of the North Esk River. coli.

This year’s conference program included two technology presen- tations presented in session 3. Dr Antonio Castillo of ThermoFisher opened by demonstrating how acoustic focussing can dramatically increase the throughput and efficiency of flow cytometry, and how this presents advantages to identifying rare events in microbial populations. Then Jaelyne Birrell and Jeffery Hochgesand-Sunjaro of Microgenetix presented platforms available for identification and

Peppers Seaport Hotel, Launceston. Venue for the 2016 Bi-State subtyping of bacterial isolates, including MALDI-TOF and Rep-PCR. conference. Two presentations rounded off the opening day of the conference, fi The Bi-State Conference was jointly organised by the ASM VIC the rst given by Associate Professor Joe Tucci of La Trobe Uni- Branch and the ASM Food Microbiology SIG. The combination of versity. Associate Professor Tucci discussed the application of these energetic working parties resulted in the compilation of as novel anti-bacterial agents, presenting a promis- an excellent and diverse scientific program for the meeting. The ing alternative to antibiotics for treating epithelial tissue infections. presentations broached a wide range of microbiology areas includ- Professor Tom Ross then discussed the fascinating application of ing clinical, diagnostic, public health, virology, veterinary, environ- bacteria to produce synthetic sandstone through cementation of mental, food and aquaculture. In total 16 oral presentations were sand grains. This biological process has the potential to revolu- delivered, and the 2016 conference also saw the inclusion of tionise building materials and has drawn attention as a possible fi a poster session with 15 posters presented over the course of the mechanism to prevent deserti cation. 2 days. Between sessions attendees had the opportunity to network and The opening presentation of the conference was delivered by visit the trade displays, with an excellent lunch and other refresh- Professor Jodie McVernon of the Doherty Institute and University ments provided at the venue. That evening attendees had the of Melbourne, giving an engaging insight into the use of historical option of joining their colleagues at the Hotel Grand Chancellor data to create models to predict the efficacy of vaccination pro- for dinner. grams directed towards pertussis. There was a strong representa- Day 2 was opened by Professor Barbara Nowak of the University of tion of the application of next-generation sequencing technologies Tasmania. A world expert on amoebic gill disease, Professor Nowak to microbiological research throughout the conference, and Asso- presented data on some of the important microbial pathogens of ciate Professor Ashley Franks of La Trobe University presented significance to mariculture. Dr Jaclyn Pearson of the Doherty research from his group on its use in high resolution profiling of Institute and University of Melbourne then presented insights into rhizosphere microbial communities. how enteropathogenic E. coli can modulate the host cell response The second session on day 1 started with an informative presen- to infection by shutting down various signalling pathways. Dr Scott tation by Angelina Jackson of the Royal Hobart Hospital describing Chandry of the CSIRO then demonstrated the power of metage- their experiences with MALDI-TOF in a clinical setting. Professor nomics in understanding microbial communities, in this case how John Bowman of the University of Tasmania then showed how it can be utilised to understand contamination patterns in food metagenomic profiling of meat spoilage organisms could be used production systems. This valuable information is helping to dra- to predict growth rates and shelf-life of vacuum packed meats. The matically improve food safety and our knowledge on how patho- Chief Scientist of Dairy Food Safety Australia, Deon Mahoney, then genic and spoilage organisms contaminate foods.

36 10.1071/MA17022 MICROBIOLOGY AUSTRALIA * MARCH 2017 ASM Affairs

that while new strategy and policy development endeavours are shaped by scientific knowledge, the final outcome may not fully reflect the original information base.

Dr Scott Chandry, CSIRO.

The following session included presentations on human public health and animal health. Dr Deborah Williamson, Deputy Director of the Microbiological Diagnostic Unit Public Health Laboratory, Dr Heather Haines, Department of Health and Human Services, Victoria. gave a riveting demonstration of how whole genome sequencing applied to public health outbreaks for rapid and effective response. The 2016 Bi-State Conference saw the introduction of poster Of particular note Dr Willamson showed how this technology can presentation sessions, which included 15 poster presentations detect otherwise unknown outbreaks occurring in various jurisdic- drawn from a wide variety of microbiological areas, and a poster tions, showcasing how this technology is revolutionising the field prize for the Best Student presentation, proudly sponsored by the of epidemiology. Veterinary Epidemiologist Dr Kevin Ellard then ASM Victorian Branch, and awarded to Tamsyn Stanborough. described an outbreak of herpesvirus which infected Pacific Tamsyn is currently undertaking her PhD through the University oysters cultivated in Tasmania, and demonstrated how this can of Tasmania but is based at the CSIRO in Victoria. Tamsyn presented Brocho- have important repercussions for market sustainability. some of her research characterising the sortase enzyme of thrix thermosphacta, which has an important enzymatic role in anchoring membrane-bound proteins.

Dr Deborah Williamson, Deputy Director of the Microbiological Diag- nostic Unit.

The final session of the conference included two presentations on communicable disease control and public health policy. Associate Professor Mark Veitch of the Tasmanian Department of Health and Human Services described the current structures in place in Australia governing communicable disease control, and highlight- From left to right: Winner of the Best Student Poster Presentation, Tamsyn Stanborough, CSIRO; Karena Waller; Ed Fox. ed the discussion over the past 30 years as to whether Australia should have its own single ‘Centre for Disease Control’ institution. The Organising Committee would also like to thank our very Dr Heather Haines of the Victorian Department of Health and generous conference sponsors, which included The Australian Human Services closed the scientific program by discussing food Society for Microbiology, ThermoFisher Scientific, Microgenetix, safety policy and regulation. In particular Dr Haines highlighted Sysmex and Becton Dickinson.

MICROBIOLOGY AUSTRALIA * MARCH 2017 37 ASM Affairs

Culture Media Special Interest Group (SIG)

Peter Traynor, MASM, MAIMS

National Convenor, Culture Media SIG Australian Society for Microbiology, Inc.

The Culture Media Special Interest Group (SIG) of the Australian 2012. In 2014–2015, the second edition of the food and water Society for Microbiology was formed in 1991 by a group of inter- guidelines were released, incorporating and recognising many of ested individuals after an upsurge in interest in the issue of media the relevant changes to the Australian Standards in food and water quality and the appearance that no common standards or consen- microbiology that had occurred over the intervening years, and the sus existed in this area in Australia. Increased interest, especially release of the international Standard ISO 11133. amongst medical microbiologists, in what was being done, or The Food and Water guidelines inclusion of both food and water should be done, by way of assuring the quality of microbiological media was the inspiration behind the decision of ISO committee media made the issue contentious. TC34/SC9 to include water media in the relevant ISO 11133 stan- The National Association of Testing Authorities (NATA) Australia, dard (currently in press), and parts of the International Standard were amongst those seeking guidance in the area of Media Quality have been heavily influenced by its Australian predecessor ...from Control, being in the position of accrediting microbiology labora- which we can draw great pride. Other citations of the ASM Guide- tories in the fields of biological testing and medical testing. They lines include the UK NHS Guidance documents, ISO Standards, found little in the way of consistency and knew of no locally NATA documents, and peer-reviewed papers in publications in- applicable guidelines on which to base their assessments and cluding the Lancet. recommendations. Recent activities A working party of the Culture Media SIG developed a set of Apart from the updates to the Guidelines, the SIG has run sessions guidelines ‘Guidelines for Assuring Quality of Medical Microbio- at recent national scientific meetings, including a workshop at the logical Culture Media’ which were approved in September 1996. 2016 meeting in Perth. The workshop was well attended, and This document was widely used over the following years and was covered the changes to the second edition of the ASM Food and acknowledged as a valuable resource by microbiologists in medical Water Guidelines, the NATA documents in this area, and ISO11133. as well as food, water, and pharmaceutical laboratories. Feedback from the attendees was extremely positive, with the The SIG’s Guidelines for Assuring Quality of Food and Water main request that we repeat the workshop again on the east coast Microbiological Media, and Guidelines for Assuring Quality of sometime in the near future. Solid Media used in Australia for the Cultivation of Medically Important Mycobacteria, both first released in 2004, also form an Your role in the SIG integral role in accreditation and certification to relevant ISO Membership of the SIG is open to all in accordance with the ASM standards. The Medical Mycology Guidelines were finally released By-Laws. We welcome all and any feedback on the existing Guide- in 2012 after a long gestation period, and the second edition of the lines at any time; any editing matters of improvement, where medical microbiological media guidelines were also released in needed, can be incorporated into the next editions.

38 10.1071/MA17013 MICROBIOLOGY AUSTRALIA * MARCH 2017 ASM Affairs

FT-035 Food Microbiology, Standards Australia Committee

Peter Traynor, MASM, MAIMS locally with an Australian Standard number (usually AS5013 series

FT-035 ASM representative number), cover pages, and the Australian appendices. It’s therefore Australian Society for Microbiology, Inc. important that, when working locally to an ISO Standard, that the Australian version is obtained to ensure compliance to the neces- FT-035 Food Microbiology Australian Standards Committee meets sary Australian variations where applicable. twice a year to review those Australian Standards (AS) that are due for review, or when a new Standardhas been proposed and requires For a full list of current Standards please use AS5013 as a search work. As reported for FT020 Water Microbiology Australian Stan- option at http://infostore.saiglobal.com/store/Portal.aspx?publish- 1 dards Committee , the committee membership includes volunteer er=AS. representatives from a diverse range of sources and institutions. 1 Whilst the ASM has one seat at the FT-035 table, many of those As per the FT020 report , when a draft Standard is released for representing other institutions are also ASM members. Those with public comment, it is critical that users of the Standard avail expertise in food microbiology related to dairy, poultry, livestock, themselves to be involved in the review process. If you, or your public health, culture media, quality assurance are amongst those laboratory, are applying AS5013 series Australian Standards in your who currently work on these Standards. There is a high degree of establishment, then it is important that your laboratory is (a) aware integration of current Australian Standards into the International that changes are being proposed, and (b) willing to ensure that the Standards Organisation (ISO) published Standards, as part of changes being proposed are sensible and understood. Therefore, it the global harmonisation of published Standards. Whilst the is critical that you, or your laboratory manager, are on the Standards local committee is unable to change the contents of a given ISO Australia mailing list for alerts for public comments on Standards Standard, there is involvement through Standards Australia in the under review. Please see https://sapc.standards.org.au/sapc/public/ writing of those ISO Standards, by way of membership of the listOpenCommentingPublication.action for more information on Working Groups that are responsible for individual ISO Standards how to make comments on draft Australian Standards. related to food microbiology, and in the Technical Committees responsible for the overall group of ISO Standards for food 2 microbiology . References Where there are needs for local variations to the ISO Standard to 1. Woodward, R. (2016) FT020 Water Microbiology Australian Standards Committee. Microbiol. Aust. 37, 50. doi:10.1071/MA16016 meet Australian requirements, these can be found as an additional 2. International Standards Organisation (ISO). http://www.iso.org/iso/home/store/ appendix to the ISO Standard. The document is then published catalogue_tc/catalogue_tc_browse.htm?commid=47920 Mycobacterium Special Interest Group (MSIG)

Lisa Shephard (MSIG Convenor)

c/o SA Pathology, Mycobacterium Reference Laboratory, Frome Road, Adelaide, SA 5000, Australia Tel: +61 8 8222 3220, Email: [email protected]

In Q4 of 2016, eight laboratories participated in the MSIG-RCPA 2017. The meeting provides members with a forum to discuss Quality Assurance Program. Laboratories will receive results and a specialised scientific information. The Agenda will include Regional discussion will be held at the next MSIG conference in 2017. Reports, an update from the National Tuberculosis Advisory Com- mittee and topical issues. The next MSIG conference will be hosted by the Mycobacterium Reference Laboratory, LabPLUS and held at the Auckland City The MSIG will also be hosting a workshop at the ASM in Hobart, Hospital in New Zealand. The meeting will be held 23–24 March Tasmania. The workshop will be held on 2 July 2017.

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17014 39 ASM Affairs

Food Microbiology Special Interest Group (SIG)

The ASM Food Microbiology Special Interest Group (SIG) had micro presentations to the program. The use of advanced tech- a busy 2016 with a number of events covering a range of food nologies to food microbiology-related areas was a feature of many microbiology-related topics. Together with the Victorian Branch, of the presentations, with Dr Scott Chandry of the CSIRO and we enjoyed three events over the course of the year and discussed Professor John Bowman of The University of Tasmania showing everything from the microbiology of Australian fermented foods, how metagenomics has revolutionised understanding and control- food spoilage and safety, public health epidemiology of foodborne ling food spoilage in production chains. Dr Deborah Williamson infections and outbreaks, to food policy and legislation. of the Microbiological Diagnostic Unit again demonstrated how Next Generation Sequencing has advanced microbiology and The first event, ‘Food contamination, outbreaks and impacts to epidemiology, allowing real-time surveillance of foodborne out- industry’ discussed some of the high-profile foodborne disease breaks and enabling rapid responses with improved public health outbreaks and contaminations that made headlines in Australia. outcomes. Dr Williamson also demonstrated how these new Dr Narelle Fegan, a Team Leader in CSIRO’s Food Safety and approaches are allowing unparalleled capacity to detect outbreaks Stability Group, provided an in-depth analysis of an infamous at a national level. Recent outbreaks of Listeria monocytogenes E. coli outbreak linked to fermented meats, which was instrumental and Shiga toxigenic Escherichia coli were discussed by Dairy Food in impacting food manufacture best practise and as Narelle pointed Safety Victoria’s Chief Scientist, Deon Mahoney. The conference out the repercussions of which were still being dealt with some also included a fascinating overview of a viral outbreak of disease 20 years after the outbreak. Dr Heather Haines then presented in Pacific Oysters by Dr Kevin Ellard. The complexities of food a review of a outbreak linked to sun-dried tomatoes. policy and legislation were also discussed by Dr Heather Haines, This was particularly interesting as this was a highly unusual food- with insights into the underlying processes that feed into this pathogen combination, and highlights the complexity of identify- important area. ing and responding to unusual foodborne outbreaks. Dr Anne Astin led the audience through the complex series of events that un- The year was rounded out for the SIG with an evening celebrating folded during the WPC 80 Clostridium contamination incident that Australian winemaking and the microbiology which underlies its occurred in 2013 and showed how when such events happen to production. Dr Kate Howell of The University of Melbourne intro- large businesses this can even have a knock-on effect to a country’s duced us to terroir and how the combination of environmental economy. Finally Nectaria Tzimourtas, a Senior Public Health conditions and the choice of yeast can impart the characteristics Officer within the Communicable Disease Prevention and Control of a wine. Matt Aulich of Blood Moon Wines then gave us a Unit at the Department of Health and Human Services, reported on fascinating insight into winemaking using wild yeast varieties, a recent emotive outbreak linked to the consumption of unpas- and how these new artisanal varieties are growing in popularity. teurised bath milk. The wealth of experience in the room also led to an engaging panel session after the talks which discussed the The SIG is looking forward to more exciting events in 2017, and complexities of food production and how a concerted effort by a with the introduction of webinar streaming we will be able to number of key stakeholders is necessary to help ensure the safest widen the audience of events to all members. ASM members food supply for consumers. who would like to join the Food Microbiology SIG can do so by The SIG then worked with the 2016 Bi-State Conference emailing [email protected] to keep up to date on events and Organising Committee to include a number of excellent food receive the SIG newsletters.

Access to Microbiology Australia Online early articles: Articles appear on the Microbiology Australia website (http:// microbiology.publish.csiro.au/) when authors have approved the pdf of their article. Completed issues: Register at http://microbiology.publish.csiro.au/ to receive notification that an issue is complete. You will get an email showing the titles and abstracts of the completed issue. You will have one click access to any article or the whole issue. Print issue: ASM members are welcome to receive the print version of Microbiology Australia without charge. To receive the print version you need to notify the ASM National Office (http:// www.theasm.org.au/).

40 10.1071/MA17015 MICROBIOLOGY AUSTRALIA * MARCH 2017 ASM Affairs

Vale Andrew Butcher

Not long after this in 2006, whilst still remaining on the branch committee, he became the Scientific Program Chair on the Local Organising Committee for ASM National Scientific Meeting in Adelaide in 2007. His organisational skills and contribution were immeasurable, resulting in yet another very successful National meeting in Adelaide.

Andrew had a keen interest in parasitology. Earlier in his career, under the supervision of Professor David Grove, Andrew under- By Paul Sideris took his PhD studies of an unusual helminth infection associated with the consumption of helicid land snails (small white land snails) in patients on the Yorke Peninsula. This work culminated in the naming of a new species of intestinal trematode worm, Brachy- It is with great sadness that I convey to you the passing of Dr Andrew laima cribbi, which reported the first human infections with this Butcher. genus of trematode.

Andrew passed away peacefully on December 8 after suffering from It wasn’t any surprise then when Andrew also took on the National the debilitating illness, MND. Despite the decline in his health over ASM Convenor of the Parasitology and Tropical Medicine SIG role. the last few years and dealing with the passing of Wendy, his wife, He held this position for 6 years, from 2006 to 2012, during which last year, Andrew remained very positive and continued to do as time he managed to instigate visits from a number of international much as he could to catch up and celebrate life with friends, family speakers to Australia, particularly world renowned Parasitologist and colleagues at every opportunity. His outlook on life was Lyn Garcia. incredible right to the end. Andrew continued to contribute to ASM right up until his retire- fi Andrew was involved and made a signi cant contribution to ASM ment by remaining on the ASM SA NT Branch Committee until 2013 throughout most of his working life, across a 20-year time period. and remained a current member of ASM. The following summarises Andrew’s life with ASM. He contributed not only to the ongoing life of the organisation but Andrew was involved in the society since 1993 and had numerous instigated and helped to provide invaluable learning experiences roles and responsibilities both within the SA NT Branch and at a for ASM members and the wider Microbiology community. He was National Level. In 1993 he first joined the SA NT Branch as a not only a regular presenter but also organised many parasitology committee member and remained on the committee right up until workshops and seminars at local, national and international ASM his retirement in 2013. meetings over many years.

Soon after joining the committee Andrew became heavily involved Andrew was also an adjunct lecturer in the School of Pharmacy and in the branch. He was the branch Scientific Meetings Convenor Medical Sciences at the University of South Australia. His involve- from 1994 to 1997 and from 1999 to 2002. From 1994 Andrew also ment included providing material and presenting and supervising took on the role of State ASM Convenor of the Special Interest specialist lectures and practical workshops in Parasitology to Lab- Group in Parasitology and Tropical Medicine, a position he held oratory Medicine students. In doing so he gave many students the until 2010. benefit of his many years of experience in diagnostic microbiology and encouraged them to pursue careers in this area and be involved From 1995 to 1997 he was part of the Local Organising Committee in ASM. Andrew received an ASM Distinguished Service Award in for the ASM Adelaide 1997 National Scientific Meeting and the 2015 for his immeasurable contribution to ASM and Microbiology as Social Program Chairperson. As many remember it was a hugely a whole over many years. successful national meeting with a fantastic social program. If that wasn’t enough he accepted a nomination of ASM SA NT Branch Andrew will be greatly missed by his many work and ASM friends Committee Chair and was in that position from 2002 to 2004. and colleagues.

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17017 41 ASM Affairs

Alison Vickery and the typing of staphylococci in Australia

Alison took over the role of ‘phage typing in the 1970s just as new strains of methicillin-resistant S. aureus (MRSA) began appearing in hospitals along the east coast of Australia. She continued to propagate the International Phage Set for use in Australia, liased with other workers in the field around the world, introduced experimental phages, and studied lysogeny and its consequences in S. aureus. For many years she provided a typing service for most of the hospitals of Australia, including the typing of strains isolated By Richard Benn in a major AGAR survey1.

Alison Vickery, who died in December 2016, played an important By the 1990s, tracking the movement of MRSA became a major task ’ role in the bacteriophage typing of Staphylococcus aureus in this of our hospital s Infection Control Unit. It was much assisted by fi country. The technique was introduced by Phyllis Rountree in the phage typing and con rmed the major role of intensive care units in 2 1950s at Royal Prince Alfred Hospital, where it was initially used to the spread of infection . Newer typing methods have now replaced identify a particularly virulent strain of S. aureus (phage type 80/81) phage typing in the study of staphylococcal infection but I believe in the neonatal nursery. that most of what we know about the epidemiology of the disease was determined by the continued efforts of Alison and her collea- The work continued in the 1960s when Phyllis Rountree and Molly gues during the latter part of the 20th century. Baird (nee Pegler) studied the epidemiology of staphylococcal infections in the hospital’s surgical units. Much of what is known about the hospital transmission of staphylococcal infection stems from this era. It revealed that most surgical wound infections are References ’ ’ caused by the patient s (rather than the attendants ) strain of 1. Vickery, A.M. et al. (1992) ‘Phage typing of clinically significant methicillin resistant S. aureus and it noted the importance of staff, particularly those Staphylococcus aureus in Australia. AGAR data. et al with chronic skin disease, in the spread of S. aureus within the 2. Barakate, M.S. . (2000) An epidemiological survey of methicillin-resistant Staphylococcus aureus in a tertiary referral hospital. J. Hosp. Infect. 44,19–26. ward. doi:10.1053/jhin.1999.0635 Future issues of Microbiology Australia May 2017: Industrial Microbiology Guest Editors: Ian Macreadie and Ipek Kurtbo¨ke September 2017: Early Careers Research November 2017: Public Health Microbiology Guest Editors: Helen Smith and Ian Macreadie March 2018: Environmental Microbiomes Guest Editor: Linda Blackall May 2018: Guest Editor: David Smith September 2018: Tick-borne diseases/pathogens Guest Editor: Stephen Graves November 2018: Biodeterioration and detrimental effects of microorganisms Guest Editor: Ipek Kurtbo¨ke

42 10.1071/MA17018 MICROBIOLOGY AUSTRALIA * MARCH 2017 ASM Affairs

Vale Jennifer Taplin BSc (21/4/1929–21/10/2016)

Typhimurium. To master this technique, she raised funds to return to Colindale. Back in Melbourne she struggled to cope with the huge demand for results.

Jenny’s work was extremely careful and her record-keeping was meticulous. She was swift to spot, and follow up, the unusual. In 1977 this led to her recognising the onset, among much else, of the slowly evolving national outbreak of Salmonella Bredeney among young children. From this dramatic episode, rose the By Joc Forsyth National Salmonella Surveillance Scheme to which Jenny’s knowl- edge contributed. Jenny’s fun side was demonstrated by her role in the jocular video produced to celebrate the 1977 furore. Here Jenny was born in Ballarat. Soon after, her parents moved to a 300 she played both a condescending state premier and a salmonella acre property at Millbrook. She was educated at home until, aged dancing the Petronella. seven, she was able to cycle to the local, one-teacher, State School. In this environment she acquired a permanent love of nature. In Her ingenuity also was tested in a solo assignment to isolate ’ 1941, as a shy only child, she was sent to board at the Hermitage salmonellas in Tuvalu. Here she had to raid the island s garbage ’ CEGGS. Her father’s death just before her final examinations led tip for glassware which couldn t bring on the aircraft with her. to her failure. She repeated the matriculation with success, but While she did little undergraduate teaching, she often spoke at ’ had to attend a boy s college to study science subjects. meetings of microbiologists and food technologists. She estab-

When she applied to the University of Melbourne to study biology lished excellent and trusted relations with colleagues across the she was guided towards bacteriology. Jenny, while a sensitive country as well as within the Victorian health department. When fi person, had an attractive and gentle personality with a quirky sense she retired in 1989, experts in the eld came from across the of humour. Specially in her final year she made life-long friendships country to speak at her festschrift. among her classmates. Janet Clarke Hall, where she resided, also In retirement she took up acting as a volunteer guide at the earned her lasting loyalty. Melbourne Zoo. She was also able to travel widely often combining When the final examinations were over she joined the Public Health this with -watching. Alas, all too soon, she was struck down by a Laboratory (PHL) within the Department of Bacteriology. Here she severe stroke that followed surgery. She showed amazing courage was intimidated by the idiosyncratic but meticulous Miss Merrified, in this shocking adversity which finally rendered her unable to live and, initially, was terrified of Dr Michael Wilson, the assistant independently. She then suffered years of increasing pain and director. In 1953, she and some colleagues found their pictures dependency without complaint and with remarkable cheerfulness. in the press during the national outbreak of typhoid and salmo- She died swiftly after a respiratory infection. nellosis stemming from imported coconut. For the next two years All the time there was another, private, side to Jenny Taplin. She was she followed her mother, first to Sydney and then to England, always a devoted member of the Toorak Uniting Church. Her family working in bacteriology in both places. She returned to re-join the circle was small but on this she bestowed support and affection. PHL (later re-named the Microbiological Diagnostic Unit) and here Her devoted care for her mother was exemplary. Her supportive she remained. Whenever possible, however, she managed to travel sympathy for friends in distress was outstanding. Her general to improve and extend her expertise. In 1962 she worked at niceness and exceptional kindness in times of trouble were noted Colindale with Dr Pat Carpenter. Back in Melbourne she worked, in the tributes which flowed in after her death. primarily, on enteric pathogens. PHL was the regional reference laboratory for the phage-typing of Salmonella Typhi. Jenny built She was a marvellous, able and conscientious colleague with a up an encyclopaedic knowledge of the evolution of typhoid in gorgeous laugh who shall live long in the memories of all who Australia. She was also sent the phages for the typing of Salmonella knew her.

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17019 43 ASM Affairs

Vale Sue Dixon

greatly influenced future approaches to investigations of outbreaks of food borne disease and the methods adopted by regulatory authorities and the Australian dairy industry to prevent Salmonella contamination of dried milk products. This development was especially important in meeting food safety requirements for valuable export markets for Australian dried milk products. As a consequence of the national media attention to this investigation Sue became widely reported as ‘Salmonella Sue’! By George Davey, AM The discovery in 1977 of S. bredeney in infant formula and the detection around the same time of S. adelaide in calcium caseinate Friends and colleagues of Sue Dixon were saddened to hear of her in a range of dried dairy based food products such as invalid diet passing in August 2016 after a short illness. supplements and slimming diets highlighted the importance of a national Salmonella typing system. This led to a collaboration Sue was born on 10 January 1928 in Malvern, Adelaide. After between the SRL, MDU, the Commonwealth Department of Health graduating from Unley High School she commenced her career and the Australian Society for Microbiology (ASM) in establishing a in microbiology as a laboratory assistant cleaning test tubes at the National Salmonella Surveillance Scheme (NSSS). Sue played a key then recently established Institute of Medical and Veterinary Sci- role in the development of the NSSS which has been a major tool in ence (IMVS). Sue was awarded a cadetship by the IMVS to study at the epidemiological investigations of salmonellosis and early warn- Adelaide University where she graduated with a BSc in 1949. From ing to health authorities of food-borne disease from Salmonella. 1949 to 1952 Sue worked as a bacteriologist at the IMVS and then resigned to start a family. After rejoining the IMVS in 1960 Sue Sue was an active member of the ASM where she served on the assumed responsibility for the National Salmonella Reference Membership Committee from 1979 to 1981 and the working party Centre established by her mentor and good friend the eminent to investigate the establishment of a Fellowship category (FASM) Dr Nancy Atkinson. From 1967 until her retirement in 1983 Sue was from 1982 to 1983. She was also President of the South Australian the head of the Salmonella Reference Laboratory (SRL) and Food Branch of ASM from 1979 to 1981. In 1983 Sue was elected to Hygiene Laboratory at the IMVS. Honorary Life Membership of ASM.

Sue was one of a number of formidable and influential female Sue was also active in the Australian Institute of Food Science and Australian microbiologists of her generation who played leading Technology (AIFST). In 1974 she participated in the inaugural roles in transforming the profession of food microbiology in this AIFST/CSIRO/UNSW Specialist course for the food Industry: country. Her contemporaries included Margaret Dick (Kraft Foods, Food-borne microorganisms of public health significance, where Melbourne), Jenny Taplin (MDU The University of Melbourne), Dr with good humour and diplomacy she taught the teachers some Barbara Keogh (CSIRO Dairy Research Laboratories, Melbourne), basics in food microbiological techniques. For subsequent courses Professor Nancy Millis (The University of Melbourne) and Dr Phillis (1976 and 1979) Sue co-authored with George Davey chapters Rountree (Royal Prince Alfred Hospital, Sydney). in Food-borne microorganisms of public health significance (the ‘Green Book’) on serological techniques for the identification of In 1977 Sue and her colleague Jenny Taplin at the Microbiological Salmonella species. Diagnostic Unit (MDU) identified infant formula as the likely cause of a widespread national outbreak of infant from Prior to the establishment of Food Standards Australia New Zealand Salmonella bredeney. However, it was only through Sue’s forensic (FSANZ) and its predecessor organisation the National Food and innovative approach to microbiological analysis of foods that Authority (NFA), microbiological specifications for foods were the source was ultimately confirmed and traced to contaminated developed by the National Health and Medical Research Council milk powder ingredient, which was something no other Australian (NHMRC), Food Microbiology Sub-committee. Sue served on this food testing laboratory was able to achieve. Her work in this area committee from around 1979 to 1983.

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Sue played a significant role in helping both government and Apart from the important part Sue played in developing national industry Australian food microbiological testing laboratories to microbiological food standards, improving laboratory testing improve testing methodologies and performance. She was a methodologies and procedures, assisting in the epidemiological member of Standards Australia Committee FT/4 Methods for the investigations of food-borne disease and surveillance of Salmonel- Microbiological Analysis of Foods from around 1976 to 1983 and a la, Sue was a role model and mentor to many young aspiring food member of the National Association of Testing Authorities (NATA) microbiologists. She was always willing to offer her guidance and Biological Testing Advisory Committee from 1982 to 1987. wisdom with humour and empathy. Sue is sadly missed. Vale Joan Faoagali

Hospital. Here Joan was responsible for a busy department in a hospital with a large population of immunocompromised patients creating an endless supply of interesting microbiology. Addition- ally, the hospital had a steady stream of antibiotic resistant organ- isms, including MRSA, which was the subject of a memorable expose by ‘60 Minutes’. Joan was filmed rubbing her finger along a dusty hospital surface saying ‘and this will grow MRSA’. Fortunately one of Joan’s key loves was infection control and she developed and published several key interventions against resistant bacteria.

Joan had a great love of education and training. She spent 6 years as The following is an edited version of a self-written eulogy distrib- examiner in Microbiology for the RCPA. She was also Queensland uted to mourners at Joan Faoagali’s funeral in Brisbane on 7 representative for the RCPA for close to a decade. At her funeral, January 2017, plus additional personal comments from David RCPA President Michael Harrison gave a glowing tribute to Joan’s Paterson. huge voluntary workload for the college. Additionally, Michael Joan Faoagali is remembered by many microbiologists as a Director Nolan (formerly Chief Scientist at RBH) gave a heartfelt eulogy of Microbiology at Royal Brisbane Hospital from 1985 to 2006 and representing the feelings shared by a large number of scientists then Princess Alexandra Hospital from 2006. Born in New Zealand with whom Joan worked. in 1940 as Joan Wilson, Joan married her first husband, Malaki Joan was the first successful ‘multitasker’ I ever met. In the days Faoagali in 1964. After graduating with her medical degree from when microbiology ‘sign-outs’ were initials on a printed report, Otago University and then undertaking her junior training in Joan would bring a swathe of reports to Grand Rounds where she Invercargill, in 1968 her young family travelled to Samoa by ‘ would sign-out plus make insightful comments during the meeting. boat’. Joan soon realised that an unmet need in Samoa was She had 7 children so it was no wonder that her Saturday morning pathology so she returned to New Zealand in 1969 to undertake ‘desk clean-up’/research time was accompanied by a child or two. pathology/microbiology training. By 1974, Joan had been Joan published more than 100 peer-reviewed papers and as appointed as Director of Microbiology at Christchurch Hospital. recently as November 2016 attended scientific meetings (despite Unfortunately Joan’s husband, Malaki, developed a malignant having breast cancer metastasised to bone and liver). Her funeral paravertebral tumour and he died in 1978. In 1985, Joan and her was standing room only, and a wonderful mix of Samoan spirit, second husband Jim Gwynne travelled with her family to take up family emotion and reflection by more than 50 professional an appointment as Director of Microbiology at Royal Brisbane colleagues.

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17020 45 ASM Affairs

ASM Science Meets Business report

Four ASM representatives, Prof. Liz Hartland, Prof. Liz Harry, Prof. research innovation, we rank 81st when it comes to the translation Enzo Palombo and A/Prof. Dena Lyras, attended Science & Tech- of that research by business. Several other countries, including nology Australia’s second annual ‘Science Meets Business’ meeting the UK and Switzerland, produce greater research outputs than held on 24 October 2016 in Melbourne. Around 200 leaders from Australia with proportionally smaller GDP spends. The reasons for research organisations, the private sector and government this disparity were explored. Former Chief Scientist Ian Chubb attended this meeting which examined cultural, policy and eco- suggested that there is a cultural problem in Australia and that nomic barriers to better collaboration between the STEMM re- teaching, science and research are not valued by Australians. He search sector and the corporate sector. also believes that this thinking is embedded in our education system, which is skewed against excellence in STEMM studies. Keynote speaker Joanna Batstone, who is the head of IBM Research NHMRC CEO Prof. Anne Kelso commented that young people in Australia and Chief Technology Officer for IBM Australia and New our sector need to be immersed in commercialisation, which will Zealand, began the day with an inspiring keynote address which allow them to develop an understanding of what is required to included success stories of corporate investment in research and successfully navigate this sector. Funding challenges were also development. The Assistant Minister for Industry, Innovation and discussed from the perspective of all sectors represented at this Science, Hon. Craig Laundy MP, and Shadow Minister for Innova- meeting. tion, Industry and Science, Senator Kim Carr, gave the Government and Opposition perspectives. Some key messages were that a Science and Technology Australia and the annual Science meets greater culture of collaboration is needed and that new initiatives Business meeting must be applauded for pushing forward the will see the Government itself is moving away from being a conversations that must happen between science and business. regulator to becoming a business partner. Discussion panels on Although there is a long way to go, the discussions held at this the nexus between Government, research and industry, entre- meeting brought together the ideas and people required to high- preneurship, emerging technologies and looking for the next light this neglected interactive space. Scientists are essential to big idea stimulated very lively discussions and featured a wealth successful and forward-thinking business development, particular- of experience from leaders in industry, science, universities and ly in this new age of climate change and reduced resource sustain- other agencies. ability and energy production. A culture of better communication The statistics presented early in the day were striking: while we and collaboration is essential, which is fostered through initiatives are the 10th most innovative nation in the world with respect to such as the Science meets Business forum.

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ASM @ Science Alive! 2016

Stephen Kidd (Chair ASM SA/NT)

In Adelaide and as a partnership with National Science Week, there we had a dark room built into the corner of the booth for viewing is a major event, the science fair – Science Alive!. This was held in glow-in-the-dark bacteria. Amazingly, in 2016 we were fortunate 2016 at the Adelaide Showgrounds across three days, Friday 5 enough to have a 9 m  3 m booth! Incredibly, we filled this space August, Saturday 6 August and Sunday 7 August. This exhibits with displays, people and visitors with little room or time to breathe various aspects of science and technology to the community (but certainly hoping again to have this size booth in 2017). (Friday being a careers day for high school students and Saturday In 2016 we established an active and enthusiastic sub-committee and Sunday is opened to the public). The Australian Society for tasked with organising and running the ASM booth at Science Microbiology (ASM – SA/NT branch) had a booth with various Alive! – Stephen Kidd, Paul Sideris, Heather Rickard, Steph activities and displays for School students. We had volunteers Lamont-Friedrich, Jennifer Singh, Helena Ward, Alistair Standish from across the SA microbiology community and this included and Brian Ho. We had displays that included glow-in-the-dark microbiology undergraduate and postgraduate students. In the bacteria, a microscope with ‘healthy’ poo (and other things), agar planning, the setting up and across the extremely busy three days, plates with dirty hands (these were big agar plates with a child’s a wonderful and enthusiastic time was had displaying and describ- dirty hand print and then after they had washed it and then after ing the importance and joys of microbiology. further cleaning – there is a story there), agar plates with samples Science Alive! has now been going for 11 years. This year the from the soil, animals (chicken and dog) putting their paws onto an Science Alive! program included over 60 organisations exhibiting agar plate, reading glasses, and then clinical plates (sealed) showing science, engineering and technology. There were guest presenters – haemolysis, antibiotic resistance/sensitivity (disk diffusion), and Reuben Merman (The Surfing Scientist – from the ABC) and plates with fungi. We also had ‘give-aways’, chiefly little key-rings Professor Rob Morrison and Dr Deane Hutton (of The Curiosity with a spray bottle of antimicrobial wash that we had made and Show,1972–1990). Tickets were free for children under 18 years printed with: STOP THE SPREAD. Then there was huge excitement of age. A busy and incredible atmosphere filled the two main at an area we had with large model pictures of the different shapes pavilions of the Adelaide Showgrounds. In 2016 there was again an of bacteria (rods, cocci...), and some play-dough so children could impressive and wonderful attendance (record numbers came in make their play-dough models of bacteria and put them into a petri 2016, creeping up to close to 30 000 people who attended over the dish; we sealed and labelled it and they could take it home (e.g. they three days). could use some gold play-dough to make some Golden Staph and we labelled it ‘Staphylococcus aureus’). In 2015, the ASM SA/NT branch was involved in Science Alive! for the first time. In 2015 exhibitors’ booths and the booth spaces were It was a huge effort to have the booth brightly and professionally arranged around requirements and a cost. For community and displayed and resourced and with the number of volunteers re- not-for-profit organisations there is a 3 m  3 m booth. For a 6 m  quired throughout the three days. This year there were additional 3 m booth the cost is $950 (+GST). For commercial/government responsibilities and costs for the booth holders – for instance, exhibitors the booths are 6 m  3 m ($1950 + GST) or 9 m  3m we were required to provide our own public liability insurance. ($2700 + GST). We originally arranged for a (free) 3 m  3 m booth It was a big, big effort for the team, but richly worthwhile to and were fortunate enough to finally have a 6 m  3 m booth. Other engage students and the public and bring a real awareness of costs are incurred for additions to the booth. For instance, in 2016 microbiology!

MICROBIOLOGY AUSTRALIA * MARCH 2017 10.1071/MA17023 47 ASM Affairs

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