Ecology of Filoviruses: the Link to Bats

Transcription

1 Photo: T&P Herron Ecology of Filoviruses: the Link to Bats Jonathan S. Towner, PhD Viral Special Pathogens Branch, CDC, Atlanta, USA Department of Pathology, College of Veterinary Medicine, University of Georgia, USA The findings and conclusions in this presentation are from the author and do not necessarily represent the views of the CDC

2 Complete genome analysis of Filoviruses 1 Ravn Ravn Kenya DRC Marburgviruses Marburg DRC DRC 1999 Ozolin Zimbabwe c Angola 2005 Musoka Kenya 1980 Popp Uganda % nt 1 1 Zaire 1995 Zaire 1976 Ebolaviruses Bundibugyo 2007 Cote d Ivoire 1994 Sudan 2000 Reston % nt 39% nt Towner et al., PLoS Pathogens, 21 Nov 2008

3 Marburg viruses: The Roadmap Discovery of the Egyptian rousette bat (Rousettus aegypiacus) as a natural reservoir Longitudinal studies of virus persistence in R. aegypiacus Experimental studies Modeling Ebola viruses: Where are we now? Lessons learned: What can we apply to the Ebola virus reservoir search?

4 Where do Ebola- and Marburgviruses reside in nature? How are these viruses transmitted from nature to humans? The approach so far has often been to initiate ecological investigations soon after the resolution of the human component of the outbreak based upon the whereabouts and activities of the index case Unfortunately, the index case is often deceased and/or unknown Almost all of the recent Ebola outbreaks in Gabon and Republic of Congo have been traced to direct contact with infected bush meat, namely non-human primates How are non-human primates becoming infected?

5 Expectations for a virus reservoir host: Find consistent evidence of infection in population (virus, antibodies) Sufficient viral loads and shedding to maintain virus circulation in population Remain clinically healthy (population level) Host species natural history is supportive of virus persistence in the population

6

7 100 Max likelihood of VP35 frag (~300nt) isolated from MHF cases At least 9 virus introductions into the human population during the Durba outbreak Contrasts all previous known filovirus outbreaks up to that time which were the likely result of single introductions substitutions/site 82 rycger67aug poppger67aug 02DRC99apr26 2 musken80jan Bausch et al., 2006 NEJM 355:909 ravken87aug10 09DRC99may26 hogzim75feb12 ozozim75feb19 cruzim75feb26 03DRC99apr30 04DRC99may01 10DRC99aug06 11DRC99aug18 19DRC00feb23 32DRC00aug13 34DRC00aug DRC99apr12 06DRC99may01 07DRC99may DRC99may09 14DRC00jan22 30DRC00aug05 23DRC00mar DRC00apr04 31DRC00aug11 33DRC00aug27 05DRC99may08 12DRC00jan08 13DRC00jan15 17DRC00feb02 16DRC00feb11 20DRC00feb12 15DRC00feb DRC00feb14 21DRC00feb24 22DRC00may05 26DRC00may20 27DRC00jul03 29DRC00jul14 28DRC00jul10 25DRC00apr

8 Ecological study of outbreak in Durba, DRC

9 Ecological study of outbreak in Durba, DRC Mine eventually flooded and all transmission stopped Lost opportunity to study virus ecology Swanepoel et al., 2007, Emerg Infect Dis

10 Kitaka Mine (4 cases) Lead and gold mine July cases 2 identified retrospectively Epidemiologically linked September case Epidemiologically unrelated

11 Ravn virus All Marburg complete genome 02Uganda 2007sep 09DRC 1999may Rav Kenya1987aug 04DRC 1999may 03DRC 1999apr 32DRC 2000aug 19DRC 2000feb 34DRC 2000aug 07DRC 1999may 06DRC 1999may 14DRC 2000jan 30DRC 2000aug 01DRC 1999apr 24DRC 2000apr 23DRC 2000mar 29DRC 2000jul 27DRC 2000jul 26DRC 2000may 17DRC 2000feb 16DRC 2000feb 12DRC 2000jan 20DRC 2000feb 21DRC 2000feb 22DRC 2000may 18DRC 2000feb 13DRC 2000jan 28DRC 2000jul 05DRC 1999may 15DRC 2000feb 33DRC 2000aug 25DRC 2000apr 01Uganda 2007jul Ozo Zimbabwe 1975feb Uganda 07 Human Sequences (21% diverse) Marburg virus 50 changes 09Angola 2005apr 08Angola 2005apr 04Angola 2005mar 07Angola 2005apr 05Angola 2005mar 02Angola 2005mar 01Angola 2005feb 06Angola 2005apr 03Angola 2005mar 11Angola 2005may 10Angola 2005apr Pop Germany1967aug 02DRC 1999apr Mus Kenya 1980jan

12 Blood Liver Spleen Heart Kidney Lung Reproductive CNS Photo Chris Black-WHO - multiple aliquots of tissue - one set of instruments/bat - all instruments treated in Lysol and bleach prior to next use

13 Summary of Q-RT-PCR positive bats (>800 tested) SPB log # Field # RT-PCR? Species Sex Status Yes R. aegyptiacus F Juvenile R. aegyptiacus F R. aegyptiacus M Yes R. aegyptiacus F R. aegyptiacus F Pregnant** R. aegyptiacus F R. aegyptiacus M R. aegyptiacus M Scrotal R. aegyptiacus M R. aegyptiacus F Pregnant** R. aegyptiacus F Juvenile Yes R. aegyptiacus F Juvenile R. aegyptiacus F w/pup (neg) R. aegyptiacus F Pregnant** Yes R. aegyptiacus M Juvenile Yes R. aegyptiacus M Juvenile R. aegyptiacus F Yes R. aegyptiacus F R. aegyptiacus F Juvenile Hipposideros caffer F R. aegyptiacus M Scrotal R. aegyptiacus F Pregnant** Yes R. aegyptiacus F R. aegyptiacus M Juvenile Total positive R. aegyptiacus 22 Total positive H. caffer 1 ** corresponding placentas tested negative

14 Uganda Bat Sequences concatenated NP and VP35 frags (~700bp) Marburg virus Maximum Parsimony 1 change Ravn virus 01k_ a_ a_ a_ a_ a_ a_ a_ a_ a_ a_ a_ d_ k_ Bat 44* 02U Bat 188* Bat 276 Bat 288 Bat 328 Bat 1013 Bat 782 Bat 982* 01U Bat 772 Bat d_ d_ d_ d_ d_ Bat 331* Bat 371* Bat 427 Bat d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ d_ z_ g g g g_ d_ Virus Isolate Virus Isolate Virus Isolate Virus Isolate Virus Isolate Bats- Aug 07 Bats- May 08 Diversity suggests a long association with a reservoir host

15 Geographic distribution of Rousettus aegyptiacus (Egyptian Fruit bat) Geographic distribution encompasses the location of all known Marburg outbreaks Reproductive capacity combined with the large colony sizes (>100,000) predicts large metapopulations In tropical Africa, reproduces twice a year % of adult females pregnant ~Birthing: Feb and Aug ~Breeding: May and Nov Deposited in nursery areas whereupon mothers feed with partially digested fruit By 3 months capable of free flight and at 6 months mom is pregnant again!

16 Python Cave (2 cases total) Dutch tourist visits cave July 2008 Became ill and died in Netherlands US case (tourist from Colorado) identified retrospectively Visited cave December 2007 Recovered

17 Kitaka Mine 5 virus isolates Sequences matched miners 5.1% level of active infection PCR+: 31/611 Python Cave 7 virus isolates Sequences matched Dutch tourist 2.5% level of active infection PCR+: 40/1582 Towner, Amman, Sealy et al Isolation of Genetically Diverse Marburg Viruses from Egyptian Fruit Bats. PLoS Pathogens 5(7) e Amman, Carroll, Reed, et al. (2012) Seasonal Pulses of Marburg Virus Circulation in Juvenile Rousettus aegyptiacus Bats Coincide with Periods of Increased Risk of Human Infection. PLoS Pathog 8(10): e

18 Seasonal pulses is not new: Influenza virus maintenance driven by influxes of juvenile birds

19 Do these periods of increased active infection levels among juvenile bats predict times of greater risk of spillover to humans? Photo: Bob Swanepoel

20 Percent Levels of PCR+ Juveniles 18.0 Seasons of Increased Human Risk? Birthing season Birthing season % PCR+ overall 54/65 (83.1%) p=< % PCR+ overall Adult Active infection Collection Dec Active infection Birthing Season Peak % active infection Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month of Historical Marburg Human Infection Monkeys Tourists , 1980, 1987, 2007, 2008 Miners , 1995, 1996, 1997, 1998, 1999, 2000, 2007

21 Target on their back: Unintended consequences at Kitaka mine

22 Target on their back: Unintended consequences at Kitaka mine 2012 Outbreak of Marburg virus in Ibanda, a large town near Kitaka mine Rousette bats had re-populated the mine <5% of previous levels 13% (53/400) of all bats were PCR + for marburgvirus 9 isolates

23 What do we know from experimental infection studies of bats?

24 What do we know from experimental infection studies of bats?

25 What do we know from experimental infection studies of bats? Virus found in tissues 2-9 days No gross pathology No virus detected in urine, feces or oral secretions Animals sero-convert around day 10 Animals inoculated IP and SQ with 10^5 TCID50

26 What do we know from experimental infection studies of bats?

27 Modeling Filovirus persistence in bats Used field based and experimental studies to develop and parameterize an SEIR model to explore: Critical community size (minimum population size) Incubation time Birth pulse : annual or biannual More synchronous the birth pulses require longer incubation periods (Peel et al., 2013)

28 Modeling Marburgvirus persistence in Rousettus aegyptiacus Results were that infection persisted for 25 years in 500 simulations provided: Critical community size is > 20,000 bats (near 100% when over 40,000 bats) Incubation time 21 days, but not 7 days Birth pulse : biannual

29 Summary of tropical African animals tested for evidence of Ebola Zaire, Yambuku 76 Nzara 76 Zaire Zaire 95 I. Coast Central African Republic 99 Total Mammals Chiroptera Mega Micro Rodents Insectivores Carnivores Primates Birds Reptiles Total vertebrates Arthropods ,843 30,161 Test: Virus isolation 0/2475 0/499 0/1664 0/30,657 0/1642 0/242 0/37,179 Pourrut et al., 2005 Microbes and Infection 7:

30 Ecological niche modeling

31 Ecological niche modeling 2004 Not granular enough 2014

32 Ebola Zaire reservoir search in Gabon : Response to repeated, distinct spillovers to NHPs Animals tested Bats 679 Birds 222 Small vertebrates 129 total 1030 Eric M. Leroy et al., Fruit bats as reservoirs of Ebola virus Nature 438, (1 December 2005)

33 Ebola Zaire reservoir search in Gabon Hypsignathus monstrosus PCR+ 4/21 IgG+ 4/17 Epomops franqueti PCR+ 5/117 IgG+ 8/117 Only known PCR data to date, no additional published reports Home ranges overlap outbreak locations Ebola virus never isolated from a bat Myonycteris torquata PCR+ 4/141 IgG+ 4/58 Eric M. Leroy et al., Fruit bats as reservoirs of Ebola virus Nature 438, (1 December 2005)

34 Serological evidence (only) of Ebola in Ghana bats (Hayman et al 2012) Species that previously tested positive, Leroy et al., 2005

35 2014 Ebola outbreak within home ranges of previously identified fruit bats Hypsignathus monstrosus Myonycteris torquata Epomops franqueti

36 Ebola Zaire reservoir search in Guinea 2014 Distribution of Mops condylurus Mops condylurus No PCR+ bats found No linkage to large mammal die-off Antibody evidence inconclusive Home range doesn t overlap previous Ebola outbreaks Secondary host? Peridomestic Source: IUCN Red List

37

38 Spillover can be lethal even in bats Only dead bats positive for Lloviu virus positive

39 Lessons learned: What could we apply to the Ebola reservoir search? For Ebola virus, there is no epidemiologic linkage to a restricted biome (e.g. cave/mine) For Marburg virus, the number of actively infected (PCR+) individuals is low (2-5%), suggesting the need to test 100s of animals of any single species Africa is home to >250 bat species (~25% of global bat diversity): You can do the math Reservoir animals can have mild disease but otherwise remain clinically healthy There may be seasons of increased levels of active infection in juveniles linkage to birth pulses (most African fruit bats in tropics have biannual birth pulses

40 Thank you