Laboratory techniques in rabies

Edited by

Charles E. Rupprecht LYSSA LLC

Atlanta, Georgia, USA

Anthony R. Fooks

Animal and Plant Health Agency Addlestone, Surrey, United Kingdom

Bernadette Abela-Ridder Department of Control of Neglected Tropical Diseases

World Health Organization Geneva, Switzerland

techniques in rabies

Fifth edition

Volume 1

Volume 1

Edited by

Charles E. Rupprecht LYSSA LLC Atlanta, Georgia, USA

Anthony R. Fooks

Animal and Plant Health Agency Addlestone, Surrey, United Kingdom

Bernadette Abela-Ridder

Department of Control of Neglected Tropical Diseases World Health Organization

Geneva, Switzerland

© World Health Organization 2018

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Preface v

List of abbreviations and acronyms used in this manualvi

Part 1. General considerations

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human rabies

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Part 2. Detection of virus

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ultrastructural studies and research Part 3. Demonstration of antigens

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of lyssavirus antigens

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the detection of rabies virus peptides Part 4. Demonstration of viral antibodies

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determination of rabies virus neutralizing antibodies

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Appendix 1 List of contributors

Appendix 2 WHO collaborating centres on rabies, neurovirology, viral zoonoses

and zoonoses control

Laboratory techniques in rabies

Foreword

)RU PRUH WKDQ \HDUV KXPDQV KDYH OLYHG LQ IHDU RI D ELWH IURP D UDELG DQLPDOVRPXFKVRWKDWWKHĺUVWZULWWHQDFFRXQWRIUDELHVLQWKHUGFHQWXU\}%&

VHW WKH SHQDOW\ IRU DQ RZQHUpV GRJ ELWLQJ DQRWKHU LQGLYLGXDO DW qWZRWKLUGV RI D PLQHRIVLOYHUrRUDERXWDKDOIGD\pVZRUN7RGD\RXUIRFXVLVPRUHRQSUHYHQWLQJ rabies and advocating for its elimination, rather than imposing penalties, and our XQGHUVWDQGLQJRIWKHYLUXVKDVJUHDWO\LPSURYHGVLQFHWKHUGFHQWXU\%&

7KH)RRGDQG$JULFXOWXUH2UJDQL]DWLRQRIWKH8QLWHG1DWLRQV)$2WKH:RUOG Organisation for Animal Health (OIE) and the World Health Organization (WHO) KDYHSULRULWL]HGDFWLRQDJDLQVWUDELHVDQGWRJHWKHUZLWK0HPEHUFRXQWULHVKDYH VHWDJRDORI]HURUDELHVGHDWKVE\'LDJQRVWLFVDUHFUXFLDOLQDWWDLQLQJWKLV JRDO

New laboratory techniques and advancements in science have yielded better GLDJQRVWLFWHFKQLTXHVDQGFRQWUROVWUDWHJLHVWRDLGWKHPRUHWKDQELOOLRQSHRSOH PDLQO\ FKLOGUHQ LQ $VLD DQG $IULFD ZKR DUH WKUHDWHQHG E\ WKH YLUXV HYHU\ GD\

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No diagnostic tests are available to detect the rabies virus before the onset of FOLQLFDOGLVHDVHDQGIXUWKHUUHVHDUFKRQGLDJQRVWLFWHFKQLTXHVLQWKHĺHOGRIUDELHV LVWKHUHIRUHSDUDPRXQW7KHLPSDFWRIVXLWDEOHODERUDWRU\FDSDFLW\RQVXUYHLOODQFH DQGHOLPLQDWLRQRIWKHGLVHDVHZRUOGZLGHLVHYLGHQW

7KH 2,(pV 0DQXDO RI GLDJQRVWLF WHVWV DQG YDFFLQHV IRU WHUUHVWULDO DQLPDOV provides internationally agreed standards for the production and control of vali GDWHGYHWHULQDU\GLDJQRVWLFPHWKRGVDQGYDFFLQHVIRUXVHLQDQLPDOV7KHIRXUWK HGLWLRQRI:+2pV/DERUDWRU\WHFKQLTXHVLQUDELHVKDVEHHQDJXLGLQJUHIHUHQFH IRUPDQ\UDELHVODERUDWRULHV7KHĺUVWHGLWLRQVWDWHGWKDWqUDELHVUHVHDUFK LVIDUIURPVWDWLFrDQGVLQFHLWVSXEOLFDWLRQPRUHWKDQ\HDUVDJR2,(DQG:+2 KDYHZRUNHGWRHYDOXDWHVXEVHTXHQWDGYDQFHPHQWVLQODERUDWRU\WHFKQLTXHVLQ UDELHV7KLVĺIWKHGLWLRQSURYLGHVLQVLJKWLQWRYDOLGDWHGPHWKRGVUHFRPPHQGHGIRU XVHLQGLDJQRVWLFODERUDWRULHVEXWLWDOVRLQFOXGHVUHVHDUFK:KLOHQRWFXUUHQWO\

applicable to all settings, these research methods may stimulate the development RILPSURYHGWHFKQLTXHVIRUGLDJQRVLVRIUDELHVLQWKHIXWXUH,PSURYHGGLDJQRVWLFV will strengthen surveillance of the disease, leading to enhanced control of rabies ZKHUHLWLVPRVWQHHGHG

Laboratory techniques in rabies

Preface

5DELHVKDVDQHQRUPRXVLPSDFWRQERWKDJULFXOWXUHDQGFRQVHUYDWLRQELRORJ\EXW LWVJUHDWHVWEXUGHQLVXQGHQLDEO\RQSXEOLFKHDOWK$VVXFKURXWLQHPHWKRGVIRUUDSLG ULVN DVVHVVPHQW DIWHU KXPDQ H[SRVXUHV WR UDELHV DV ZHOO DV DSSOLFDWLRQV IRU ODERUD WRU\EDVHGVXUYHLOODQFHSURGXFWLRQRIELRORJLFDOVDQGPDQDJHPHQWRIWKLVLQIHFWLRXV GLVHDVHDUHFULWLFDO*LYHQLWVPDQGDWHWRLPSURYHKXPDQKHDOWKDQGFRQWUROGLVHDVH DPRQJLWV0HPEHU6WDWHV:+2KDVOHGWKHSURGXFWLRQRIWKLVĺIWKHGLWLRQRILaboratory techniques in rabies

'XULQJWKHPRUHWKDQ\HDUVWKDWKDYHHODSVHGVLQFHWKHĺUVWHGLWLRQZDVSXEOLVKHG methods of viral diagnosis, characterization of pathogens and production of biologicals KDYHDGYDQFHG$WWKDWWLPHRQO\DVLQJOHHWLRORJLFDODJHQWZDVUHFRJQL]HGDVFDXVLQJ UDELHV'HWHFWLRQRI1HJULERGLHVZDVWKHVWDQGDUGIRUGLDJQRVLV1HUYHWLVVXHEDVHG YDFFLQHVZHUHWKHQRUP&RPELQDWLRQXVHRIYDFFLQHVDQGUDELHVLPPXQRJOREXOLQVLQ KXPDQSURSK\OD[LVZDVQRWVWDQGDUG*OREDOHOLPLQDWLRQRIFDQLQHUDELHVZDVPHUHO\D GUHDP5DELHVLQZLOGOLIHZDVPDQDJHGYLDSRSXODWLRQUHGXFWLRQ$OORIWKDWKDVFKDQJHG IRUWKHEHWWHU

In the ensuing decades, further advancements in detection, prevention and control of lyssaviruses have been monitored by regular meetings of WHO experts, international UHVHDUFKJURXSVDQGFRXQWULHVLQZKLFKUDELHVLVHQGHPLF7KHVHFRQGHGLWLRQRIWKH PDQXDOZDVSXEOLVKHGLQWKHWKLUGLQDQGWKHIRXUWKLQ7KHODWH0DUWLQ .DSODQDQG+LODU\.RSURZVNLZHUHLQVWUXPHQWDOLQHGLWLQJWKHSUHYLRXVHGLWLRQVDVZDV LQSXW RQ WKH IRXUWK HGLWLRQ E\ )UDQÄRLV;DYLHU 0HVOLQ QRZ UHWLUHG IURP :+2 ,QLWLDO SODQVIRUSUHSDUDWLRQRIWKLVHGLWLRQZHUHPDGHLQDQGLWVFRQWHQWVZHUHGLVFXVVHG DWWKH:+2([SHUWPHHWLQJRQUDELHV%DQJNRN7KDLODQGDQGPRGLĺHGLQUHVSRQVH

7KLVĺIWKHGLWLRQRILaboratory techniques in rabiesFRQWDLQVGHWDLOHGFKDSWHUV ZULWWHQE\PRUHWKDQDXWKRUVIURP$IULFDWKH$PHULFDVDQG(XUDVLD7KHWH[WZDV SHHU UHYLHZHG E\ 'U 0DWWKLDV 6FKQHOO +HDG RI WKH :+2 &ROODERUDWLQJ &HQWUH IRU Neurovirology; Professor Thiravat Hemachudha, Head of the WHO Collaborating Center IRU5HVHDUFKDQG7UDLQLQJRQ9LUDO=RRQRVHVDQG'U$VHID'HUHVVD7HDP/HDGHURI

=RRQRVHV 5HVHDUFK DW WKH (WKLRSLDQ 3XEOLF +HDOWK ,QVWLWXWH 7KH} PDQXDO IRFXVHV RQ the basic methods for detection of lyssavirus antigens, antibodies and nucleic acids and the relevance of their use under different operating conditions, from the basic to WKHDGYDQFHG7KHFKDSWHUVRQROGHUOHVVVHQVLWLYHWHFKQLTXHVXVHGWRGHWHFW1HJUL bodies have been removed, as have those chapters on methods of vaccine production given the progress made in the commercial use of tissue culture products in human and YHWHULQDU\PHGLFLQH5HFRPPHQGDWLRQVIRUWKHSUHSDUDWLRQVRIDQWLERGLHVE\KRPROR gous or heterologous production have been replaced by newer methods in an effort to promote a next generation of less expensive and more readily available immunoglobu OLQVLQWKHIXWXUH2WKHUEDVLFFKDSWHUVKDYHEHHQUHWDLQHGDQGXSGDWHGDQGPRUHWKDQD GR]HQDGGHG(DFKRIWKHSURWRFROVGHVFULEHGDUHSUHVFULSWLYHDQGVKRXOGEHIROORZHG SRLQWE\SRLQWLQWKHODERUDWRU\

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Part 1. General considerations

Chapter 1

Introduction

$VLPSOLĺHGHDUO\WKFHQWXU\YLHZRIWKHKLVWRULFDOUDELHVODERUDWRU\PD\KDYH been predisposed towards a linear, passive role of sample receipt and diagnosis RQO\7RGD\WKHLGHDOUROHRIWKHPRGHUQUDELHVODERUDWRU\LVPRUHFRPSOH[DQG PXOWLGLPHQVLRQDO7DEOH:KLOHVRPHODERUDWRULHVPD\SHUIRUPRQHRURQO\

a few core functions, others conduct much more diverse activities, especially the reference laboratories of the World Organisation for Animal Health (OIE) and the :RUOG +HDOWK 2UJDQL]DWLRQ :+2 0RUHRYHU EHFDXVH UDELHV LV D IXQGDPHQWDO disease of nature, the laboratory should be engaged not only in diagnostics per se but also in actively maintaining a central role in the assessment, planning and investigation of cases of rabies in humans, domestic animals and wildlife locally and abroad as part of routine prevention and control using a One Health approach )LJ7KLVFULWLFDOUHVSRQVLELOLW\IRULQYROYHPHQWLQKXPDQYHWHULQDU\DQGHQYL URQPHQWDOKHDOWKKDVJXLGHGWKHSURGXFWLRQRIWKLVĺIWKHGLWLRQRILaboratory tech- niques in rabies and informed the practical approach requested by countries in which canine rabies is endemic to support the goal of eliminating human deaths IURPGRJPHGLDWHGUDELHVE\

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Diagnosis Primary analytical antemortem and postmortem testing of specimens from suspect animals and humans

Discovery ,GHQWLĺFDWLRQRIQHZO\VVDYLUXVVSHFLHVUHVHUYRLUVYDULDQWVHWF

Education 3UHDQDO\WLFDOGHĺQLWLRQRIFULWLFDOHOHPHQWVWRSURYLGHUVRIURXWLQHVDPSOHFROOHFWLRQVWRUDJH VKLSSLQJHWF

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Production Preparation of animal and human biologicals for detection or prophylaxis 5HIHUHQFH ,QDXJXUDWLRQRIDFHQWUDOL]HGQDWLRQDOODERUDWRU\RUJOREDO2,(:+2QHWZRUN

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Training Tutorials and updates of current or novel methods to local, national, regional and global laboratorians

Typing 8WLOL]DWLRQRIPHWKRGVWRFKDUDFWHUL]HELRGLYHUVLW\RIYLUDOYDULDQWVSK\ORJURXSVHWF QA, quality assurance; QC, quality control; OIE, World Organisation for Animal Health; WHO, World Health Organization 7DEOH0XOWLSOLFLW\RIUROHVDQGUHVSRQVLELOLWLHVRIWKHUDELHVODERUDWRU\LQWKHVWFHQWXU\

At a minimum, all rabies laboratories should be capacitated to perform diagnosis XVLQJ WKH qJROG VWDQGDUGr GLUHFW ĻXRUHVFHQW DQWLERG\ WHVW ')$7 7KLV} KLJKO\

VHQVLWLYHDQGVSHFLĺFPHWKRGKDVEHHQLQURXWLQHXVHIRUPRUHWKDQ\HDUV,Q retrospect, this manual is a culmination not only of reliable laboratory techniques that have stood the test of time but also of newer protocols for detection of viral antigens, antibodies and amplicons, production of relevant biologicals, where DSSOLFDEOHDQGDSSOLHGUHVHDUFK

The current design relies on the successful outline of previous editions, starting ZLWKJHQHUDOFRQVLGHUDWLRQVDERXWWKHGLVHDVH7KLVĺIWKHGLWLRQLVVHSDUDWHGLQWR WZRYROXPHVWRUHĻHFWRSHUDWLRQDODQGUHVHDUFKIRFXVHGFRQWHQWDQGLVDYDLODEOH online only to facilitate rapid electronic updates as further advances in laboratory WHFKQLTXHVEHFRPHDYDLODEOH$VUDELHVLVFDXVHGE\QXPHURXVO\VVDYLUXVHVDQ introduction (Chapter 2) to the genus is an appropriate reminder that rabies virus is only one viral species of special concern and to promote further pathogen disco YHU\IURPDJOREDOSHUVSHFWLYH%LRVDIHW\Chapter 3) is of paramount importance WRDOOODERUDWRULDQVDQGWKHUHIRUHZDUUDQWVDVHSDUDWHFKDSWHU1RWDEO\KXPDQDQG DQLPDOGLDJQRVWLFVDOLNHPXVWRSHUDWHZLWKLQDORJLFDOVSKHUHIRUODERUDWRU\EDVHG surveillance to operate (Chapter 4+XPDQUDELHVLVXQGHUUHSRUWHGRQDOOFRQWL QHQWVDQGWKHFXUUHQWJXLGDQFHVHHNVWRDGGUHVVWKLVVKRUWFRPLQJChapters 5 and 6&OHDUO\DEDVHVXVSLFLRQRIKXPDQHQFHSKDOLWLVLVQRWVWUDLJKWIRUZDUGDQG VKRXOGEHDSSURDFKHGZLWKLQDVWUXFWXUHGIUDPHZRUNRIGLIIHUHQWLDOGLDJQRVHV⁽¹⁾ Advanced neuroimaging may be useful to consider an infectious etiology, inclu ding rabies ⁽²⁾$QWHPRUWHPGHWHFWLRQRIVSHFLĺFLPPXQHFRPSOH[HVFDQDOVREH useful in cases of paralytic rabies ⁽³⁾

Incident Investigation Sample collection

Specimen shipment LABORATORY

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As rabies is an acute progressive encephalitis, the central nervous system (CNS) LVWKHPDLQWLVVXHRILQWHUHVW:KHUHDVLQWKHSDVWWKHHQWLUHEUDLQZDVUHPRYHG typically for diagnosis in animals, this cumbersome and sometimes dangerous exercise is now largely unnecessary when a full section of the brainstem is avai lable (Chapter 76DIHDQGVHFXUHPHWKRGVDUHDOVRVXJJHVWHGIRUREWDLQLQJDQG VKLSSLQJVDPSOHVIURPWKHĺHOGIRUXVHLQEDVLFGLDJQRVLVDQGFKDUDFWHUL]DWLRQRI pathogens (Chapter 15 %H\RQG URXWLQH QHHGV IRU HQKDQFHG ODERUDWRU\EDVHG surveillance, laboratorians should also appreciate current regulatory norms for evaluation of diagnostic products available nationally or imported, human or animal vaccines, reagents used in applied research, and expectations of other future human and animal biologicals (Chapters 35 and 36

$ZLGHYDULHW\RIPHWKRGVDUHDYDLODEOHIRUXVHLQUDELHVGLDJQRVLV/\VVDYLUXV virions may be visualized directly via electron microscopy (Chapter 10 ,QIHF WLRXV}YLUXVHVDUHXVHGLQDGLYHUVLW\RISURWRFROVIURPSDWKRELRORJLFDOVWXGLHVWR WHVWVRIYDFFLQHHIĺFDF\DQGFDQEHLVRODWHGLQDQLPDOVChapter 8) or, preferably, in tissue culture (Chapter 9%H\RQGWUDGLWLRQDOGHPRQVWUDWLRQRIYLUDODQWLJHQV LQ WKH &16 E\ WKH ')$7 Chapter 11), an alternative is the direct rapid immu QRKLVWRFKHPLVWU\ WHVW '5,7 XVLQJ OLJKW PLFURVFRS\ Chapter 12 DV D VLPSOLĺ cation of its formal predecessor, traditional immunohistochemistry (Chapter 13 Antigenic typing of viral variants (Chapter 14PD\EHSHUIRUPHGXVLQJĻXRUHVFHQW RU OLJKW PLFURVFRS\ WR GLVFULPLQDWH ODERUDWRU\ VWUDLQV IURP ĺHOG LVRODWHV DQG WR GLIIHUHQWLDWHLQIHFWHGDQLPDOVDQGKXPDQVE\YDULDQWW\SH7KH')$7PD\DOVREH SHUIRUPHGXSRQIUR]HQVNLQELRSVLHVIRUGLDJQRVLVRIKXPDQUDELHVZLWKUHVXOWV available in a few hours (Chapter 156HYHUDORWKHUWHVWVChapters 16–18) have also been suggested for the detection of viral antigens, including latex agglutina tion protocols ⁽⁴⁾

,GHDOO\DVLPSOHSRLQWRIFDUHWHVWZRXOGJUHDWO\DVVLVWLQVXUYHLOODQFHDQGULVN DVVHVVPHQWV8QIRUWXQDWHO\FXUUHQWO\DYDLODEOHOLQHDUĻRZWHVWVYDU\IURPORWWR ORWDQGDUHQRWUHFRPPHQGHGIRUPDNLQJGHFLVLRQVRQSRVWH[SRVXUHSURSK\OD[LV 3(3LQKXPDQV

The detection of viral antibodies (Chapters 19–22) is useful for several purposes, LQFOXGLQJEDVLFGLDJQRVLVULVNDVVHVVPHQWVLPPXQRJHQLFLW\VWXGLHVDQGHSLGH PLRORJLFDO LQYHVWLJDWLRQV $QWLERG\ GHWHFWLRQ PD\ EH GLVFHUQHG IXQFWLRQDOO\ E\

QHXWUDOL]DWLRQRUWKURXJKELQGLQJVWXGLHVVXFKDVHQ]\PHOLQNHGLPPXQRVRUEHQW assays (ELISAs), as detailed in earlier editions of this manual and in a recent publi cation ⁽⁵⁾DUHQRWUHSHDWHGKHUHLQDVSURWRFROVKDYHQRWFKDQJHGFRQVLGHUDEO\

$GGLWLRQDOO\ RYHU WKH SDVW IHZ \HDUV D QXPEHU RI FRPPHUFLDO (/,6$ NLWV KDYH EHFRPH DYDLODEOH 5HFHQW LQWHUQDWLRQDO WHVWLQJ KDV VKRZQ JRRG UHSURGXFLEL lity among laboratories as particularly useful for immunological insights for oral vaccination programmes against rabies in wildlife ⁽⁶⁾ *LYHQ WKH RSSRUWXQLW\ IRU cell culture or ELISA formats, antibody detection in animals is no longer recom mended as state of the art and is included only as a courtesy for those countries LQZKLFKLWKDVEHHQUHWDLQHGKLVWRULFDOO\XQWLOQHZHURSWLRQVPD\EHDGRSWHG

%H\RQGGHPRQVWUDWLRQRIYLUDODQWLJHQVDQGDQWLERGLHVDZLGHYDULHW\RIPROH FXODU WHFKQLTXHV DUH DYDLODEOH WRGD\ IRU QXFOHLF DFLG DPSOLĺFDWLRQ DQG JHQHWLF comparisons (Chapters 27–34:KLOHLQWKHSDVWPDQ\RIWKHVHPROHFXODUSURWR FROV ZHUH XVHG LQ D FRQĺUPDWRU\ EDVLV WRGD\ PDQ\ DUH XVHG DV D VWDQGDORQH IRU SULPDU\ GLDJQRVLV %RWK FRQYHQWLRQDO DQG UHDOWLPH UHYHUVH WUDQVFULSWDVH

SRO\PHUDVHFKDLQUHDFWLRQ573&5SURWRFROVKDYHEHHQYDOLGDWHGWRFRYHUWKH VSHFWUXPRINQRZQO\VVDYLUXVHV6WULFWDGKHUHQFHWRTXDOLW\FRQWUROOLPLWVWKHULVN RI FURVVFRQWDPLQDWLRQ 3URWRFROV RWKHU WKDQ WKRVH JLYHQ LQ WKLV FKDSWHU KDYH also been validated, such as oligonucleotide microarrays for viral speciation ⁽⁷⁾ 6DQJDU}VHTXHQFLQJRI3&5SURGXFWVLVVWLOODXVHIXOPHWKRGWRXVHIRUGLIIHUHQWLD WLRQRIYDULDQWVLIWKHFRVWVRIHTXLSPHQWDQGUHDJHQWVDUHSURKLELWLYH1H[WJH neration sequencing may be inaccessible for many laboratories in the developing ZRUOGEXWLVDĺHOGWKDWLVH[SHFWHGWRJURZLQUHĺQHPHQWVLPSOLFLW\DQGFRVW

Several topics were not carried forward from the earlier editions, either because they are obsolete or not considered necessary given modern alterna WLYHV)RU}H[DPSOHWKHGHSHQGHQFHXSRQGHWHFWLRQRI1HJULERGLHVLVJURXQGHG LQ WKH WK FHQWXU\ DQG UHSODFHG E\ PDQ\ PRUH VHQVLWLYH DQG VSHFLĺF WHVWV WRGD\2WKHUSURWRFROVZHUHQRWLQFOXGHGLQWKLVHGLWLRQLIFRPPHUFLDONLWVFRQMX JDWHVRU}UHDJHQWVZHUHSURGXFHG)RUH[DPSOHPXOWLSOHFRPPHUFLDOFRQMXJDWHV DUH DYDLODEOH IRU WKH ')$7 KRZHYHU LQ ORFDOLWLHV ZKHUH DFFHVV WR WKHP LV PRUH OLPLWHG} O\VVDYLUXV SURWHLQV PD\ EH SXULĺHG DQG FRQMXJDWHV FDQ EH SURGXFHG LQKRXVH}⁽⁸⁾+HQFHRQHVXFKSURWRFROZDVUHWDLQHGIRUSURGXFWLRQRISRO\FORQDO DQWLVHUDLQJRDWVIRUXVHLQGLDJQRVWLFDSSOLFDWLRQVChapter 372WKHUVPDOOHU VSHFLHVVXFKDVUDEELWVFDQDOVREHXVHGIRUSURGXFWLRQDQGDGKRFFRQMXJDWLRQ RIDQWLVHUXP}⁽⁹⁾

Similarly, fewer protocols dealing with generation of other biologicals were UHWDLQHG WKDQ LQ SUHYLRXV HGLWLRQV PRVW DUH QRZ DEVHQW IURP WKH ĺIWK HGLWLRQ ,QIRUPDWLRQRQQHUYHWLVVXHEDVHGYDFFLQHVLVFOHDUO\REVROHWHDVWKH\KDYHEHHQ UHSODFHGE\PXFKVDIHUWLVVXHFXOWXUHYDFFLQHDOWHUQDWLYHVVXFKDVDYLDQRU9HUR FHOOYDFFLQHVIRUKXPDQV6LPLODUO\PXOWLSOHPRGLĺHGOLYHDWWHQXDWHGDQGUHFRP binant vaccines for use in domestic animals and wildlife have been licensed in Eurasia and North America and are no longer considered novel given their routine use ^(10–17)5HDGHUVLQWHUHVWHGLQWKHORFDOQDWLRQDOSURGXFWLRQRIVXFKWKFHQWXU\

human or veterinary biologicals should refer to the listed citations for review or FRQWDFWFRPPHUFLDOHQWLWLHVIRUWUDQVIHURIWHFKQRORJ\

In contrast to rabies vaccines, there are even more comparative needs today beyond human and equine rabies immunoglobulins for PEP in humans ⁽¹⁸⁾ 7KXV} FRQWULEXWLRQV RQ JHQHUDWLRQ RI PRQRFORQDO DQWLERGLHV LPPXQRJOREXOLQ <

DQG SODQWEDVHG DQWLERGLHV DUH LQWHQGHG WR VSXU IXUWKHU UHVHDUFK DQG GHYHORS ment on more cost–effective use of PEP in the future (Chapters 38–41 6LPL larly, alternatives to the standard National Institutes of Health test (Chapter 42) for measurement of vaccine potency continue to be sought ⁽¹⁹⁾7RGDWHSURJUHVVKDV been made for application to both veterinary (Chapter 43) and human (Chapter 44ELRORJLFDOV

7KLVĺIWKHGLWLRQRILaboratory techniques in rabies focuses on the needs of labo ratories for surveillance of rabies in humans and animals as well as that of preven WLRQDQGFRQWUROSURJUDPPHV5DELHVFRQWLQXHVWRFDUU\WKHKLJKHVWFDVHIDWDOLW\

UDWHRIDQ\LQIHFWLRXVGLVHDVH$VXQGHUVWDQGLQJRIYLUDOSDWKRELRORJ\LPSURYHV and relevant models for investigation become available, future editions may include suitable antiviral approaches for PEP and therapy, as are being deve ORSHGIRURWKHUQHJDWLYHVWUDQGHG51$YLUXVHV⁽²⁰⁾,QWKHLQWHULPHDUO\HFRQR PLFDOGHWHFWLRQFKDUDFWHUL]DWLRQDQGUHVSRQVH)LJUHPDLQWKHKDOOPDUNVIRU UHGXFLQJWKHEXUGHQRIWKLVDQFLHQW]RRQRVLV

References

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Chapter 2

Lyssaviruses

Introduction

5DELHV LV DQ DFXWH YLUDO HQFHSKDOLWLV ZLWK D FDVH IDWDOLW\ UDWH DSSUR[LPDWLQJ ZKLFK LV WKH KLJKHVW DPRQJ NQRZQ LQIHFWLRXV GLVHDVHV 7KLV ]RRQRVLV NQRZQWRKXPDQNLQGIRUPLOOHQQLDLVDVVRFLDWHGSULPDULO\ZLWKWKHELWHVRIqPDGr dogs ⁽¹⁾ 7KH (QJOLVK QDPH OLNHO\ RULJLQDWHV IURP WKH 6DQVNULW ZRUG qUDEKDVr PHDQLQJqYLROHQFHr$VLPLODUHW\PRORJ\H[LVWVLQRWKHUODQJXDJHVWKHQDPHRI WKHGLVHDVHW\SLĺHVXQFRQWUROOHGYLROHQFHDJJUHVVLRQPDGQHVVRUIUHQ]\

%HVLGHV qFODVVLFDOr UDELHV YLUXV 5$%9 D QXPEHU RI RWKHU SK\ORJHQHWLFDO O\UHODWHGYLUXVHVVHJUHJDWHLQWRWKHJHQXV^Lyssavirus as described during recent decades ⁽²⁾6RPHRIWKHVHYLUXVHVDUHDQWLJHQLFDOO\DQGVHURORJLFDOO\UHODWHGWR 5$%9ZKHUHDVRWKHUVDUHPRUHGLYHUJHQWDQGDVVXFKDUHQRWFRYHUHGIXOO\E\

commercial biologicals for rabies prophylaxis ^(3–5)6WLOOPRUHWKDQRIKXPDQ UDELHVFDVHVDUHFDXVHGE\LQIHFWLRQZLWK5$%9PHGLDWHGE\GRJV3RVWH[SR sure prophylaxis (PEP) virtually assures complete prevention of the disease if DGPLQLVWHUHGSURPSWO\DQGSURSHUO\DQGKDVEHHQDYDLODEOHIRURYHUDFHQWXU\

Nevertheless, rabies still causes tens of thousands of human deaths every year due to poor availability or affordability of appropriate biologicals, particularly in those areas where the demand for them is highest, such as developing countries of tropical Asia and Africa ⁽⁶⁾

As with any infectious disease, effective programmes of rabies management UHO\RQULJRURXVODERUDWRU\EDVHGVXUYHLOODQFHUREXVWGLDJQRVWLFWRROVDQGKLJKO\

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Virion structure and life cycle

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SURWHLQ0SUHVHQWEHWZHHQWKHVHWZRXQLWVFRQGHQVHVWKH1&DQGLQWHUDFWVZLWK the G protein endodomain ⁽⁷⁾

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template, and acts as a chaperone during synthesis of the N protein by forming N–P FRPSOH[HVWKDWSUHYHQWWKH1SURWHLQIURPVHOIDJJUHJDWLRQDQGELQGLQJWRFHOOXODU 51$⁽⁷⁾7KH3SURWHLQDOVRLQWHUDFWVZLWKWKHKRVWFHOOXODUWUDQVSRUWV\VWHPVVXFK DVWKHG\QHLQPRWRUFRPSOH[QXFOHRF\WRSODVPLFWUDQVSRUWHUVDQGPLFURWXEXOHV

(10–12)WRIDFLOLWDWHLQWUDFHOOXODUPRYHPHQWRIYLUDOFRPSRQHQWV)XUWKHUPRUHWKH P protein interferes with innate immunity by inhibiting different steps of the host FHOOLQWHUIHURQUHVSRQVH7KH0SURWHLQELQGVWR1&VDQGWKHF\WRSODVPLFGRPDLQ of the G protein, thereby facilitating the budding process, and also mediates such pathobiological effects as cell rounding, intracellular membrane redistribu WLRQ DQG DSRSWRVLV 7KH * SURWHLQ LV WKH RQO\ YLUDO FRPSRQHQW SUHVHQW RQ WKH YLULRQVXUIDFH7KHYLUDO*SURWHLQLVRUJDQL]HGLQWULPHUVSURWUXGLQJWKURXJKWKH YLULRQHQYHORSHDQGFRQVLVWVRIDQHQGRF\WRSODVPLFGRPDLQWUDQVPHPEUDQH GRPDLQDQGHFWRGRPDLQ7KHODWWHUPHGLDWHVELQGLQJWRKRVWFHOOUHFHSWRUVDQG induces endocytosis and fusion of viral and endosomal membranes, whereas the LQWHUQDO GRPDLQV RI WKH * SURWHLQ LQWHUDFW ZLWK WKH 0 SURWHLQ IDFLOLWDWLQJ YLULRQ PRUSKRJHQHVLV DQG EXGGLQJ $V WKH RQO\ H[WHUQDO FRPSRQHQW WKH * SURWHLQ LQGXFHVSURGXFWLRQRIYLUXVQHXWUDOL]LQJDQWLERGLHV91$DQGHOLFLWVFHOOPHGLDWHG LPPXQHUHVSRQVHV7KH/SURWHLQKDVPXOWLSOHGRPDLQVDQGSHUIRUPVWKHIXQF WLRQVUHTXLUHGIRUJHQRPHWUDQVFULSWLRQDQGUHSOLFDWLRQLQFOXGLQJ51$GHSHQGHQW 51$SRO\PHUDVHP51$pFDSSLQJHQ]\PHFDSPHWK\OWUDQVIHUDVHpSRO\$

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14)$IWHUYLULRQVELQGWRFHOOXODUUHFHSWRUVDQGHQGRF\WRVLVWKHHQGRVRPDOYHVLFOH LVWUDQVSRUWHGE\UHWURJUDGHD[RQDOĻRZGHSHQGHQWRQWKHPLFURWXEXOHQHWZRUN XVLQJG\QHLQPRWRUV7KHGHFUHDVHRIS+ZLWKLQWKHHQGRVRPHSURYRNHVFRQIRU mational changes of the G protein, which mediates fusion between the endo VRPDODQGYLUDOPHPEUDQHVDQGWKHŗVWUDQG51$JHQRPHHQFDSVLGDWHGE\WKH N protein is liberated into the cytoplasm ^{(11, 12)}7UDQVFULSWLRQDQGUHSOLFDWLRQRFFXU DWWKHQHXURQDOSHULNDU\RQ

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During their synthesis, both the nascent antigenome and the genome are encap VLGDWHGE\WKH1SURWHLQ

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Viral pathogenesis

Generally, virions are delivered into a wound via a bite or, rarely, contamination RIPXFRXVVXUIDFHVVXFKDVKLJKO\FRQFHQWUDWHGDHURVROVJHQHUDWHGLQODERUDWRU\

settings or in the unique environment of certain caves inhabited by colonies of PXOWLPLOOLRQLQGLYLGXDOEDWVLQFOXGLQJTadarida brasiliensis(19)8VLQJHLWKHUVHQVRU\

RUPRWRUQHXURQVYLUXVHVPRYHFHQWULSHWDOO\WRWKH&161HXURWURSLVPDYRLGVRU limits surveillance by the host immune system, resulting in the absence of an early DQWLERG\ UHVSRQVH 7KH GXUDWLRQ RI D QRQFOLQLFDO LQFXEDWLRQ SHULRG DYHUDJHV t}PRQWKVEXWPD\YDU\IURPDZHHNWRVHYHUDOPRQWKVRUHYHQ\HDUV^{(20, 21)} During the incubation period, rabies diagnosis cannot be performed on animal or human tissues due to the very limited viral load, uncertain location in peripheral QHUYHVDQGWKHDEVHQFHRIDGHWHFWDEOHLPPXQHUHVSRQVH

Upon delivery to the CNS, lyssaviruses disseminate rapidly and may affect QHDUO\DOOUHJLRQVRIWKH&167KHPHGXOODREORQJDWDLVWKHPDLQDUHDIURPZKLFK virus is transported to the salivary glands along innervation pathways, allowing further excretion with saliva and transmission to other susceptible hosts via a bite ⁽²²⁾7KHUHIRUHWKHPRVWUHOLDEOHGLDJQRVLVRIUDELHVFDQEHGRQHGXULQJWKH clinical period of the disease (or after death) on CNS tissues; the brainstem is the PRVWLPSRUWDQWUHJLRQIRUGLDJQRVWLFWHVWV9LUXVFDQEHVSUHDGFHQWULIXJDOO\IURP the CNS to peripheral tissues also via innervation pathways, allowing antemortem GHWHFWLRQ RI DQWLJHQV DQG QXFOHLF DFLGV LQ KDLU IROOLFOHV E\ D VNLQ ELRSV\ DW WKH QDSH RI WKH QHFN 6XFK GHSRVLWLRQ RI SHULSKHUDO WLVVXH PD\ SRVH D KD]DUG IRU scavenging mammals when they consume carcasses of rabid animals (23) and in human recipients of tissue or organ transplantation ^{(24, 25)}

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prolong the disease beyond a month ⁽²⁶⁾$IHZFDVHVRIUHFRYHU\IURPFOLQLFDO rabies have been documented in humans and animals ^{(6, 27)}

Phylogeny and taxonomy

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were included in the same serotype ^{(29, 30)})XUWKHUULJRURXVVWXGLHVZLWKH[WHQGHG panels of monoclonal antibodies helped to distinguish between these viruses as ZHOO DV EHWZHHQ (XURSHDQ EDW YLUXVHV WKHPVHOYHV 6XFK WHUPV DV qELRW\SHVr appeared in the taxonomic literature for a limited period of time ⁽³¹⁾+RZHYHU WKH PRVW FRPSUHKHQVLYH GLIIHUHQWLDWLRQ RI UDELHVUHODWHG YLUXVHV ZDV DFKLHYHG via sequencing and comparison of their genes ⁽³²⁾7KHVHODWWHUHIIRUWVLQJHQHUDO FRQĺUPHGDQGFODULĺHGĺQGLQJVIURPPRQRFORQDODQWLERG\VWXGLHVEXWFODVVLĺ cation switched from viral serotypes to genotypes, along with the establishment of the genus ^LyssavirusZKHUHDOOUDELHVUHODWHGYLUXVHVKDYHEHHQLQFOXGHG7KH H[LVWLQJqJHQRW\SHVrVHUYHGDVDEDVLVIRUWD[RQRP\RIO\VVDYLUXVHVIRUDOPRVW WZRGHFDGHVEXWZHUHUHĺQHGWRVDWLVI\WKHRIĺFLDOUXOHVHVWDEOLVKHGE\WKH,QWHU QDWLRQDO &RPPLWWHH RQ 7D[RQRP\ RI 9LUXVHV ,&79 ZKLFK RSHUDWHV ZLWK PRUH FRPSOH[GHĺQLWLRQVVXFKDVYLUDOVSHFLHV

Increased surveillance has led to the discovery of several novel lyssaviruses GXULQJ UHFHQW GHFDGHV $W SUHVHQW WKH JHQXV LQFOXGHV DW OHDVW HVWDEOLVKHG VSHFLHV DQG RQH YLUXV DZDLWLQJ ,&79 DVVHVVPHQW 7DEOH 'XULQJ WKH ,&79 LQWURGXFHG D YDULDQW RI ELQRPLDO YLUXV QRPHQFODWXUH⁽³³⁾ 7KLV ZLOO OLNHO\

HYROYH LQ WKH IXWXUH DV LQ WKH SUHVHQW IRUP LW PD\ FDXVH FRQIXVLRQ HJ UDELHV YLUXV>DYLUXV@EXWRabies lyssavirus>DYLUDOVSHFLHV@2IQRWHRQO\YLUXVQDPHVEXW QRWVSHFLHVQDPHVFDQEHDEEUHYLDWHG)LJ7KLVPDQXDOXVHVYLUXVQDPHV when in reference to the viruses, whereas species names are used solely in the

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L *HQHWLF GLVWDQFHV ZLWK WKH WKUHVKROG RI t QXFOHRWLGH LGHQWLW\ IRU WKHFRPSOHWH1JHQHRUtQXFOHRWLGHLGHQWLW\IRUWKHFRQFDWHQDWHG FRGLQJ UHJLRQV RI 130*/ JHQHV *OREDOO\ DOO LVRODWHV EHORQJLQJ WR the same species have higher identity values than the threshold, except WKHYLUXVHVFXUUHQWO\LQFOXGHGLQWRWKH/DJRVEDWO\VVDYLUXVVSHFLHV%DVHG RQWKHVHREVHUYDWLRQVDQGRQDSRRUFURVVQHXWUDOL]DWLRQEHWZHHQLVRODWHV from different Lagos bat virus lineages, several authors have suggested subdividing Lagos bat lyssavirus into several separate species ^{(34, 35)} (ii) Topology and consistency of phylogenetic trees, obtained with various

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(iv) Additional characteristics such as ecological properties, host and geogra phical range; pathological features are considered, whenever available ⁽⁷⁾ 0RUHJOREDOO\EDVHGRQJHQHWLFGLVWDQFHVDQGVHURORJLFDOFURVVUHDFWLYLW\WKH Lyssavirus genus has been subdivided into two main phylogroups ⁽³⁶⁾7KHGHPDU FDWLRQ ZDV EDVHG RQ SK\ORJHQHWLF UHODWLRQVKLSV ł DPLQR DFLG VHTXHQFH identity within the G ectodomain between viruses of one phylogroup and }}VHTXHQFHLGHQWLW\EHWZHHQYLUXVHVIURPGLIIHUHQWSK\ORJURXSVFURVVQHX WUDOL]DWLRQ ZLWKLQ SK\ORJURXSV EXW DEVHQFH RI FURVVQHXWUDOL]DWLRQ EHWZHHQ PHPEHUVRIGLIIHUHQWSK\ORJURXSVWKHSUHVHQFHRI.5LQWKH*RISK\ORJURXS ,YLUXVHVDQGRI'LQWKH*RISK\ORJURXS,,YLUXVHVZLWKWKHIROORZLQJLPSOLFD WLRQIRUDUHGXFHGSDWKRJHQLFLW\IRUWKHODWWHU*HQHUDOO\VHURORJLFDOFURVVUHDFWL vity correlated somewhat with genetic distances, and sequences that shared more WKDQDPLQRDFLGVHTXHQFHLGHQWLW\ZLWKLQWKHLU*HFWRGRPDLQVFURVVQHXWUD lized each other ⁽³⁶⁾

6HYHUDOO\VVDYLUXVHVFKDUDFWHUL]HGLQWKHVWFHQWXU\HJ:&%9>:HVW&DXFD VLDQEDWYLUXV@,.29>,NRPDO\VVDYLUXV@DQG//(%9>/OHLGDEDWO\VVDYLUXV@FRXOG QRWEHLQFOXGHGLQWRHLWKHURIWKHVHJURXSV3K\ORJHQHWLFDOO\DOOWKUHHYLUXVHVDUH UHODWHGEXWORQJJHQHWLFGLVWDQFHVDQGDSSDUHQWDEVHQFHRIFURVVQHXWUDOL]DWLRQ EHWZHHQ:&%9//(%9DQG,.29GRQRWDOORZSODFLQJWKHPLQWRRQHSK\ORJURXS EDVHGRQWKHH[LVWLQJGHPDUFDWLRQFULWHULD)LJ

7KH OLPLWHG VHURORJLFDO FURVVUHDFWLYLW\ EHWZHHQ O\VVDYLUXVHV IURP GLIIHUHQW SK\ORJURXSV LV LPSRUWDQW IURP D SXEOLF KHDOWK DQG YHWHULQDU\ SHUVSHFWLYH $OO FRPPHUFLDOO\DYDLODEOHUDELHVELRORJLFDOVDUHEDVHGRQ5$%9DQGHOLFLWHIĺFLHQW protection against phylogroup I lyssaviruses but not against other members of the JHQXVSDUWLFXODUO\WKRVHO\VVDYLUXVHVLQSK\ORJURXS:&%9,.29DQG//(%9}^(3,

36–38)0RUHRYHUDQWLJHQLFDQGJHQHWLFGLVWLQFWLRQVVKRXOGEHFRQVLGHUHGLQDSSOL cation of any diagnostic tests to ensure their ability to detect all divergent lyssa YLUXVHVDVGHVFULEHGLQWKHUHOHYDQWFKDSWHUVRIWKLVPDQXDO

Recognized and proposed species

Phylogroup Natural host Geographical

range Comments

Rabies lyssavirus (type species)

I %DWV&KLURSWHUD of multiple species, terrestrial mammals (predominantly Carnivora)

Terrestrial mammals – worldwide (except Australia, Antarctica and several insular territories); bats – New World only

5HVSRQVLEOHIRUPRVWFDVHV RIKXPDQUDELHVLQWKHZRUOG All currently available human and veterinary vaccine strains originate from viruses of this VSHFLHV

Australian bat lyssavirus

I Pteropodid bats (at least four species of Pteropus genus) and insectivorous bats Saccolaimus albiventris)

Australia (perhaps, with several surrounding islands)

Given limited surveillance, host range among insectivorous bats PD\EHJUHDWHU7KUHHKXPDQ FDVHVGRFXPHQWHG

European bat lyssavirus,W\SH

I Insectivorous bats (predominantly Eptesicus serotinus)

7KHPDMRUSDUWRI Europe, from Spain to WKH8NUDLQH

Given the limited surveillance in eastern Europe and Asia, may be distributed more broadly, along the reservoir species UDQJH6SLOORYHULQIHFWLRQVLQ wild and companion animals, as well as a human case have been GRFXPHQWHG

European bat lyssavirusW\SH

I Insectivorous bats (predominantly Myotis daubentonii and Myotis dasycneme)

1RUWKZHVWHUQ Europe

Two human cases have been GRFXPHQWHG

Khujand lyssavirus I Insectivorous bat Myotis mystacinus

Central Asia .QRZQE\DVLQJOHLVRODWH Given the limited surveillance in eastern Europe and Asia, may EHGLVWULEXWHGPRUHEURDGO\

No human cases have been GRFXPHQWHG

Aravan lyssavirus I Insectivorous bat Myotis blythii

Central Asia .QRZQE\DVLQJOHLVRODWH Given the limited surveillance in eastern Europe and Asia, may EHGLVWULEXWHGPRUHEURDGO\

No human cases have been GRFXPHQWHG

Bokeloh bat lyssavirus

I Insectivorous bat Myotis nattereri

Europe Known by three isolates from :HVWHUQ(XURSH1RKXPDQ FDVHVKDYHEHHQGRFXPHQWHG Irkut lyssavirus I Insectivorous bats

from Murina genus

Eastern Asia Known by three isolates, from bats and from a human who GHYHORSHGUDELHVDIWHUDEDWELWH Duvenhage

lyssavirus

I Insectivorous bats 6XE6DKDUDQ$IULFD Known by four isolates, three of which came from humans, bitten by bats, and one from a bat, of the presumably Miniopterus VSHFLHV

7DEOH9LUXVHVFXUUHQWO\LQFOXGHGLQWKHJHQXV^Lyssavirus

Recognized and proposed species

Phylogroup Natural host Geographical

range Comments

Lagos bat lyssavirus

II Pteropodid bats of several genera (Eidolon helvum, Rousettus aegyptiacus, EpomophorusVSS HWF

6XE6DKDUDQ$IULFD Includes several lineages ZLWKORQJJHQHWLFGLVWDQFHV Potentially, in the future may be VXEGLYLGHGLQWRtVHSDUDWH VSHFLHV6SLOORYHULQIHFWLRQV reported in wild and companion DQLPDOV1RKXPDQFDVHV GRFXPHQWHG

Mokola lyssavirus II 8QNQRZQ 6XE6DKDUDQ$IULFD Twice isolated from shrews, once

IURPDURGHQW7KHPDMRULW\RI other isolates were obtained from companion animals, such as cats and dogs, as the result of VSLOORYHULQIHFWLRQV7ZRKXPDQ FDVHVKDYHEHHQUHSRUWHG Shimoni bat

lyssavirus

II Insectivorous bat Hipposideros commersoni(H.

vittatus)

Kenya .QRZQE\DVLQJOHLVRODWH

Serological surveys suggest that Hipposideros commersoni (H.

vittatusLVWKHOLNHO\UHVHUYRLU No human cases have been GRFXPHQWHG

West Caucasian bat lyssavirus

Insectivorous bats from genus Miniopterus

6RXWKHDVWHUQ Europe, probably Africa

Known by a single isolate from WKH&DXFDVLDQUHJLRQ+RZHYHU serological surveys suggest WKDWWKLVRURWKHUVHURORJLFDOO\

related virus may be present in Miniopterus bats in Africa .HQ\D1RKXPDQFDVHVKDYH EHHQGRFXPHQWHG

Ikoma lyssavirus African civet

Civettictis civetta

8QLWHG5HSXEOLFRI Tanzania

.QRZQE\DVLQJOHLVRODWH7KH QDWXUDOKRVWLVTXHVWLRQDEOH Given phylogenetic relatedness to the West Caucasian bat virus, the index case in African civet PD\UHVXOWIURPDVSLOORYHU LQIHFWLRQRIEDWRULJLQ1RKXPDQ FDVHVKDYHEHHQGRFXPHQWHG Lleida bat

lyssavirus

Insectivorous bat Miniopterus schreibersii

6SDLQDQG)UDQFH

>XQSXEOLVKHG GDWD@

.QRZQE\DVLQJOHLVRODWH No human cases have been GRFXPHQWHG

Gannoruwa bat lyssavirus

I ,QGLDQĻ\LQJIR[

Pteropus medius (P. giganteus)

6UL/DQND Several isolates obtained from the single bat species in Sri /DQND*LYHQWKHKRVWUDQJH may be present in the Indian subcontinent and surrounding DUHDV1RKXPDQFDVHVKDYH EHHQGRFXPHQWHG

Taiwan bat lyssavirus

I Insectivorous bat Pipistrellus abramus

Taiwan, China .QRZQE\WZRLVRODWHV Given the host range, may be distributed broadly in eastern

$VLD1RKXPDQFDVHVKDYH EHHQGRFXPHQWHG

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Distribution, evolution and host range

Lyssaviruses are distributed globally except in Antarctica and several insular LVODQGV ,Q WKH ODWWHU LQWURGXFWLRQ RI WKH LQIHFWLRQ LV VWLOO SRVVLEOH ZLWK PLJUDWRU\

bats, or via human activity as happened with the introduction of rabid dogs followed by host switching to populations of mongooses (introduced by humans as well) in Grenada ⁽³⁹⁾ DQG 3XHUWR 5LFR⁽⁴⁰⁾, or the introduction of rabid ferret badgers to Taiwan ⁽⁴¹⁾

$OWKRXJK DOO ZDUPEORRGHG DQLPDOV LQFOXGLQJ ELUGV⁽⁴²⁾, are susceptible, PDPPDOVIURPWKHRUGHUV&KLURSWHUDEDWVDQG&DUQLYRUDDUHWKHPDMRUQDWXUDO reservoirs in which lyssaviruses perpetuate and evolve ⁽⁴³⁾ 7KH PDMRU GLYHUVLW\

RI O\VVDYLUXVHV LV LQ EDWV ZKHUHDV 5$%9 DV WKH PRVW HYROXWLRQDU\ VXFFHVVIXO UHSUHVHQWDWLYHRIWKHJHQXVLVKLJKO\DGDSWHGWRFLUFXODWLRQLQFDUQLYRUHV3RVVLEOH H[FHSWLRQVLQFOXGH,.29ZKLFKZDVLVRODWHGRQO\RQFHIURPDFDUQLYRUHHJDQ

$IULFDQFLYHWEXWJLYHQWKHSK\ORJHQHWLFUHODWHGQHVVRI,.29WRVXFKYLUXVHVDV :&%9DQG//(%9LWLVSRVVLEOHWKDW,.29LVDEDWYLUXV⁽⁵⁾$QRWKHUH[FHSWLRQLV 02.9ZKLFKZDVLVRODWHGVHYHUDOWLPHVIURPVKUHZVGRPHVWLFGRJVDQGFDWV EXWLWLVQRWNQRZQZKHWKHUDQ\RIWKHVHDQLPDOVVHUYHDVUHVHUYRLUVRUDFFLGHQWDO hosts of the virus ⁽⁴⁴⁾

In addition, all lyssavirus diversity has been documented in the Old World but RQO\5$%9ZDVGHWHFWHGLQWKH1HZ:RUOG,WLVUHPDUNDEOHWKDWLQWKH1HZ:RUOG EDWVRIGLIIHUHQWVSHFLHVPDLQWDLQFLUFXODWLRQRIPXOWLSOH5$%9OLQHDJHVZKHUHDV LQWKH2OG:RUOGEDWVKDUERXUDYDULHW\RIGLYHUJHQWO\VVDYLUXVHVEXWQRW5$%9 Such discrepancies compromise the commonly accepted theory that lyssaviruses HYROYHGLQEDWVDQGWKDWDWOHDVWWKHLQLWLDOHYROXWLRQWRRNSODFHLQWKH2OG:RUOG OLNHO\LQ$IULFDZKHUHWKHPDMRULW\RIGLYHUJHQWO\VVDYLUXVHVDUHSUHVHQW⁽⁴³⁾,Q}SDUWL FXODU LI ZH DFFHSW WKH HVWLPDWLRQV RI O\VVDYLUXV DJH EDVHG RQ PROHFXODU FORFN WHFKQLTXHVZKLFKLQGLFDWHWKDWPRVW5$%9OLQHDJHVHPHUJHGWHQVWRKXQGUHGVRI

\HDUVDJRWKHQWKHHQWLUHJHQXVLVQRROGHUWKDQt\HDUV^(45–47)

An increasing amount of evidence suggests that timescale estimation performed on a limited set of recently sampled sequences cannot provide realistic inferences for viruses evolving under constraints of purifying selec WLRQ}⁽⁴⁸⁾7KLVLVH[DFWO\WKHFDVHIRUO\VVDYLUXVHVWKDWHYROYHQHXWUDOO\XQGHUVWURQJ purifying selection constraints ⁽⁴⁹⁾,QJHQHUDOHYROXWLRQDU\UDWHVREVHUYHGLQDVHW of sequences exhibit time dependency and increase towards the present because RIWKHWUDQVLHQWPXWDWLRQV\HWWREHUHPRYHGE\SXULI\LQJVHOHFWLRQ6XEVWLWXWLRQ rates estimated during long time frames will therefore be lower systematically WKDQ WKRVH REWDLQHG GXULQJ VKRUW WLPH IUDPHV 7KLV HIIHFW LV DV VWURQJ DV RU stronger than, purifying selection, which shapes virus evolution ^{(50, 51)},QRWKHU words, timescale estimations based on substitution rates are useful only for the time frame encompassed by the sampling period, but cannot be extrapolated IRUORQJHUSHULRGVRIWLPH)RUH[DPSOHDQDOWHUQDWLYHDSSURDFKZLWKLPSURYHG substitution saturation, increased the age of coronaviruses by several orders of PDJQLWXGHFRPSDUHGZLWKSUHYLRXVPROHFXODUFORFNHVWLPDWHVDQGWKHUHVXOWLQJ timescale was comparable to that of the coronavirus reservoir hosts ⁽⁵²⁾3HUKDSV lyssaviruses should be considered more ancient pathogens that may have evolved in bats during the Cretaceous period in the territory of modern Africa (the region IURPZKLFKWKHEDWVRULJLQDWHGDQGWKDWDQDQFLHQW5$%9IROORZHGEDWGLVSHUVLRQ

WR6RXWK$PHULFDtPLOOLRQ\HDUVDJRYLDLVODQGKRSSLQJDQGLQWHUFRQWLQHQWDO ĻLJKW}⁽⁵³⁾7KLV}K\SRWKHVLV⁽⁵⁴⁾PLJKWH[SODLQDQLQGHSHQGHQWHYROXWLRQRI5$%9 in New World bats and the absence of the virus in related taxa of Old World bats HJ IURP JHQHUDEptesicus, Myotis 6XFK 1HZ :RUOG EDW YLUXVHV IXUWKHU VKLIWHG WR FDUQLYRUHV LQFOXGLQJ VNXQNV DQG UDFFRRQV ZKLFK DUH SDUWV RI WKH qLQGLJH QRXV$PHULFDQ5$%9OLQHDJHrDQGDUHZHOOGLVWLQJXLVKHGIURPDOO5$%9OLQHDJHV FLUFXODWLQJ LQ WKH 2OG :RUOG DQG IURP WKH qFRVPRSROLWDQr FDQLQH 5$%9 ZKLFK is dispersed globally, with rabid dogs accompanying human migrations during recent centuries ^{(55, 56)}

8VXDOO\ HDFK O\VVDYLUXV SK\ORJHQHWLF OLQHDJH LV DVVRFLDWHG ZLWK D VSHFLĺF KRVW7KHVHUHODWLRQVKLSVDUHEHVWVWXGLHGIRU5$%9ZKLFKLVGLVWULEXWHGJOREDOO\

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distributed bat species, such as Eptesicus fuscus (the big brown bat) and ^Tadarida brasiliensisWKH0H[LFDQIUHHWDLOHGEDWKDUERXUVHYHUDOOLQHDJHVRI5$%9^{(47, 57)}

$PRQJ ZLOG FDUQLYRUHV VNXQNV RI VHYHUDO GLIIHUHQW VSHFLHV DSSHDU KLJKO\

SHUPLVVLYHWRYDULRXV5$%9YDULDQWV,QDGGLWLRQWRWKHVRXWKFHQWUDODQG0H[LFDQ VNXQN5$%9VZKLFKDUHDVVRFLDWHGSK\ORJHQHWLFDOO\ZLWKEDWYLUXVHVWKHUHDUH VHYHUDORWKHUOLQHDJHVLHQRUWKFHQWUDOVNXQN&DOLIRUQLDVNXQNDQGRWKHUVNXQN OLQHDJHVIURP0H[LFRZKLFKEHORQJWRDqFRVPRSROLWDQr5$%9OLQHDJH6NXQNV LQ HDVWHUQ &DQDGD KDYH DFTXLUHG WKH $UFWLF 5$%9 YDULDQW^{(55, 56)} $GGLWLRQDOO\

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Conversely, Procyon lotor (the North American raccoon) seems well adapted DQG FRQVWUDLQHG RQO\ WR RQH VSHFLĺF 5$%9 YDULDQW ZKLFK LV EHOLHYHG WR KDYH HPHUJHGGXULQJWKHVLQ)ORULGD86$DQGWKHUHDIWHUKDVUDSLGO\VSUHDGDORQJ the eastern coast of North America ^{(60, 61)}

2WKHU$PHULFDQOLQHDJHVRI5$%9FLUFXODWLQJLQGRJVFR\RWHVCanis latrans) DQGVHYHUDOVSHFLHVRIIR[HVEHORQJWRWKHqFRVPRSROLWDQrJURXS0DQDJHPHQW RI FDQLQH UDELHV UHVXOWHG LQ HOLPLQDWLRQ RI GRJPHGLDWHG UDELHV LQ WKH 86$ DQG

&DQDGDDOWKRXJK5$%9VWLOOFLUFXODWHVLQVRPHSDUWVRI/DWLQ$PHULFD0RQJRRVHV LQVHYHUDO&DULEEHDQLVODQGVDFTXLUHGWKHqFRVPRSROLWDQr5$%9IURPGRJVDQG maintain its circulation ⁽⁵⁶⁾2UDOUDELHVYDFFLQDWLRQRIZLOGOLIHVLJQLĺFDQWO\UHGXFHG 5$%9FLUFXODWLRQLQWKHLUSRSXODWLRQVLQ1RUWK$PHULFD,QDGGLWLRQWRWKHUHSUH VHQWDWLYHVRIqFRVPRSROLWDQr5$%9OLQHDJH$UFWLF5$%9FLUFXODWHVDPRQJ$UFWLF foxes (Vulpes lagopus), red foxes (^{V. vulpes}DQGVWULSHGVNXQNVMephitis mephitis) in northern territories of the continent ^{(62, 63)}

,Q WKH 2OG :RUOG 5$%9 KDV EHHQ GRFXPHQWHG LQ FDUQLYRUHV RQO\ ZLWK WKH H[FHSWLRQ RI HSL]RRWLFV LQ JUHDWHU NXGX DQWHORSHV Tragelaphus strepsiceros) in southern Africa ⁽⁶⁴⁾, predominantly in canids and herpestides ⁽⁶⁵⁾7KHqFRVPR SROLWDQrOLQHDJHRI5$%9FLUFXODWHVLQGRJVDQGZLOGFDQLGVLQYDVWWHUULWRULHVRI Eurasia and Africa ^{(55, 56)},QDGGLWLRQGLVWLQFWqDXWRFKWKRQRXVrOLQHDJHVRI5$%9 FLUFXODWHLQGRJVLQVHYHUDODUHDVRI$IULFDDQGVRXWKHDVW$VLD$UFWLF5$%9FLUFX ODWHVFLUFXPSRODUO\DPRQJ$UFWLFDQGUHGIR[HV3K\ORJHQHWLFLQIHUHQFHVVXJJHVW WKDWWKH$UFWLF5$%9OLQHDJHRULJLQDWHGIURPVRFDOOHG$UFWLFOLNHOLQHDJHVZKLFK are present in the Indian subcontinent and in central and eastern Asia ^{(63, 66)} 5DFFRRQ GRJV Nyctereutes procyonoides), besides maintaining the circulation of

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