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The pathogenesis of Powassan virus in mice after airborne infection Gaunt, Roderick Allan 1972

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THE PATHOGENESIS OF POWASSAN VIRUS IN MICE AFTER AIRBORNE INFECTION by RODERICK ALLAN GAUNT B.Sc. U n i v e r s i t y o f A l b e r t a 1961 M.Sc. U n i v e r s i t y o f A l b e r t a 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department o f Microbiology We accept t h i s t h e s i s as conforming to the requi red standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1972 i In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s tudy . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copy ing o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8. Canada Date ABSTRACT THE PATHOGENESIS OF POWASSAN VIRUS IN MICE AFTER AIRBORNE INFECTION Although arboviruses u s u a l l y i n f e c t humans by b i t e s o f i n f e c t e d t i c k s or mosquitoes a c c i d e n t a l i n f e c t i o n with several agents has been encountered f o l l o w i n g i n h a l a t i o n o f v i r u s - l a d e n aerosols generated a c c i d e n t a l l y during s p i l l a g e o f material i n the l a b o r a t o r y . This p r o j e c t provides a model of a c c i d e n t a l airborne i n f e c t i o n o f man by Powassan v i r u s . The s p e c i f i c aims were: 1. to determine i f mice could be i n f e c t e d with Powassan v i r u s , a member o f the Russian spring-summer complex of t i c k - b o r n e group B arboviruses by a means which d i d not introduce the v i r u s d i r e c t l y i n t o the bloodstream; 2. to provide information on the dose-response r e l a t i o n s h i p s using various routes of i n o c u l a t i o n ; 3. to determine the pathogenesis of Powassan v i r u s i n mice a f t e r airborne i n f e c t i o n . Intranasal i n s t i l l a t i o n o f Powassan v i r u s suspensions i n t o anaes-t h e t i z e d mice induced a f a t a l i n f e c t i o n by a route which d i d not introduce v i r u s d i r e c t l y i n t o the bloodstream. Although mice developed f a t a l e n c e p h a l i t i s f o l l o w i n g i n o c u l a t i o n of Powassan v i r u s by several routes, the s m a l l e s t q u a n t i t y of v i r u s which induced an overt i n f e c t i o n was i n j e c t e d e i t h e r i n t r a c e r e b r a l l y or i n t r a v e n o u s l y . Fatal i n f e c t i o n s followed the subcutaneous i n j e c t i o n of 20 times the minimum amount o f v i r u s . Mice could not be i n f e c t e d by the i n s t i l l a t i o n of more than one m i l l i o n i n t r a c e r e b r a l doses of v i r u s d i r e c t l y i n t o the g a s t r o i n t e s t i n a l t r a c t . I n f e c t i o n by i n h a l a t i o n of aerosols r e q u i r e d about 600 times the mini-mum v i r u s dose, and i n f e c t i o n was i n i t i a t e d by the i n t r a n a s a l i n s t i l l a t i o n o f 10,000 or more minimum i n t r a c e r e b r a l doses o f v i r u s . i i The i n f e c t i v i t y o f Powassan v i r u s suspensions and aerosols decayed r a p i d l y under u l t r a v i o l e t i r r a d i a t i o n . A e r o s o l i z e d v i r u s was s l i g h t l y more s t a b l e under c o n d i t i o n s of low r e l a t i v e humidity i n c o n t r a s t to high and intermediate r e l a t i v e h u m i d i t i e s , when aerosols were aged up to f i v e hours i n the dark at 21°C. A f t e r aerosol i n f e c t i o n o f mice with Powassan v i r u s the f o l l o w i n g sequence of events occurred. Powassan v i r u s invaded and m u l t i p l i e d w i t h i n the e p i t h e l i a l c e l l s and/or macrophages of the mouse lung. Subsequently, vir u s appeared i n the blood. When the v i r u s t i t r e w i t h i n the blood a t t a i n e d approximately 3 logjQ mouse i n t r a c e r e b r a l LD 5 Q per ml. at two days post expo-sure i t appeared i n other t i s s u e s i n c l u d i n g the b r a i n where i t m u l t i p l i e d to t i t r e s as high as 9 log^Q mouse i n t r a c e r e b r a l LD^Q per gram by the seventh day. Mice died with e n c e p h a l i t i s seven to e i g h t days a f t e r aerosol i n h a l a -t i o n . Using f l u o r e s c e n t antibody techniques, i t was determined that v i r u s present i n the blood o f the mouse invaded the cuboidal e p i t h e l i u m o f the choroi d plexus and l a t e r spread e i t h e r from t h i s t i s s u e to the cerebrospinal f l u i d o r d i r e c t l y by way of the v a s c u l a r endothelium to the t i s s u e s o f the b r a i n . E l e c t r o n micrographs of nasal e p i t h e l i u m i n f e c t e d with Powassan vi r u s presented very p r e l i m i n a r y information on the p o s s i b l e morphogenesis of the v i r u s w i t h i n t h i s t i s s u e . Cytoplasmic vacuoles were observed which could serve as the s i t e f o r sy n t h e s i s of v i r u s p a r t i c l e s . TABLE OF CONTENTS Page HISTORICAL REVIEW . ... • • • 1 Introduct i o n . . . . 1 Arbovirus Group 2 Powassan Virus ... 6 Pathogenesis and Pathology o f Virus Diseases . ... 7 V i r a l Invasion o f Central Nervous System ... 9 Neural Spread o f Virus to the Central Nervous System ... 11 Hematogenous Spread o f Virus to Central Nervous System „ 15 O l f a c t o r y Spread of Virus to Central Nervous System 20 Airborne Virus I n f e c t i o n s 21 MATERIALS AND METHODS . , 27 Virus 27 Fluorescent Antibody ... 28 Routes o f I n f e c t i o n 31 ( i ) Intr a c e r e b r a l I n o c u l a t i o n 31 ( i i ) Intranasal I n s t i l l a t i o n . 32 ( i i i ) Subcutaneous Inoculation 33 ( i v ) Intravenous I n o c u l a t i o n . 33 ( v) I n t r a p e r i t o n e a l I n o c u l a t i o n 33 ( v i ) Per 0s Ino c u l a t i o n . . ... 33 ( v i i ) Aerosol I n o c u l a t i o n . 34 Aerosol Techniques 35 ( i ) Powassan Virus Decay S t u d i e s . . 37 ( i i ) C r o s s - I n f e c t i o n . 39 ( i i i ) Aerosol Exposure of Mice . 39 Histopathology ... 41 i v Page ( i ) H i s t o l o g i c a l Techniques . . . . . . 41 ( i i ) S e c t i o n i n g Techniques 41 ( i i i ) Fluorescence Microscope Techniques 42 ( i v ) E l e c t r o n Microscope Techniques 43 RESULTS 45 Virus 45 Fluorescent Antibody 45 Routes o f I n f e c t i o n 45 Intranasal I n s t i l l a t i o n 46 Aerosol I n o c u l a t i o n 47 Powassan Virus Decay Studies . . . . 47 C r o s s - I n f e c t i o n 49 Aerosol Exposure o f Mice 49 Histopathology 50 DISCUSSION 52 LITERATURE CITED 136 v LIST OF TABLES Page Table 1 A n t i g e n i c C l a s s i f i c a t i o n o f Arboviruses ACAV . . 72 Table 2 A n t i g e n i c C l a s s i f i c a t i o n o f Arboviruses ACAV 1970, 1971 . . . . . . . . 73 Table 3 Prototype S t r a i n s o f the Russian Spring-Summer Complex o f Tick-Borne Group B Arboviruses 74 Table 4 Minimum L D 5 Q of Powassan Virus i n Mice by Various Routes o f I n f e c t i o n . 75 Table 5 C r o s s - I n f e c t i o n Record , 76 Table 6 Levels of Powassan Virus i n Various Mouse Tissues A f t e r Intranasal I n s t i l l a t i o n o f Virus . 77 Table 7 Levels o f Powassan Virus i n Various Mouse Tissues A f t e r Aerosol I n o c u l a t i o n o f Virus 78 Table 8 B i o l o g i c a l Decay o f Powassan Virus Suspensions i n the Dark and Under U l t r a v i o l e t I r r a d i a t i o n 79 Table 9 B i o l o g i c a l Decay of Powassan Virus Aerosols at Low, Intermediate and High R e l a t i v e Humidities and Constant Temperature 80 Table 10 B i o l o g i c a l Decay o f Powassan Virus Aerosols i n the Dark and Under U l t r a v i o l e t I r r a d i a t i o n . . . . . . . . . 81 vi Page Plat e 13 S a g i t t a l S e c t i o n o f Powassan Virus Infected Mouse Brain a t the S i t e of Cleavage Planes Between Adjacent Portions o f the B r a i n S i x Days A f t e r Aerosol I n o c u l a t i o n Stained With Hematoxylin and:Eosjn 119 Plate 14 S a g i t t a l S e c t i o n o f Powassan Virus Infected Mouse Brain a t the S i t e o f Cleavage Planes Between Adjacent Portions o f the B r a i n S i x Days A f t e r Aerosol I n o c u l a t i o n Stained With Hematoxylin'and; Eosjn. 119 Plate 15 Cross-Section o f Powassan Virus Infected Mouse Lung Two to Three Days A f t e r Aerosol I n o c u l a t i o n Stained With FITC Conjugated Powassan Virus S p e c i f i c Rabbit Gamma G l o b u l i n 121 Pl a t e 16 Cross-Section o f Powassan Virus Infected Mouse Lung Five Days A f t e r Aerosol I n o c u l a t i o n Stained With FITC Conjugated Powassan Virus S p e c i f i c Rabbit Gamma G l o b u l i n . 121 Pla t e 17 S a g i t t a l S e c t i o n o f Powassan Virus Infected Mouse Brain Showing the C e l l s o f the Choroid Plexus of the Mouse as they Protrude Into One o f the V e n t r i c l e s of the Brain Three Days A f t e r Aerosol I n o c u l a t i o n , Stained With FITC Conjugated Powassan Virus S p e c i f i c . . . . 123 Pl a t e 18 S a g i t t a l S e c t i o n o f Powassan Virus Infected C e l l s of the Choroid Plexus o f the Mouse Three Days A f t e r Aerosol I n o c u l a t i o n Stained with FITC Conjugated Powassan Virus S p e c i f i c Rabbit Gamma G l o b u l i n . . . . . . . 123 i x Page. Plat e 19 S a g i t t a l Section o f Powassan Virus Infected C e l l s o f the Choroid Plexus o f the Mouse Three Days A f t e r Aerosol I n o c u l a t i o n Stained With FITC Conjugated Powassan Virus S p e c i f i c Rabbit Gamma Gl o b u l i n 125 Plate 20 S a g i t t a l S e c t i o n o f Powassan Virus Infected C e l l s o f the Choroid Plexus o f the Mouse Three Days A f t e r Aerosol I n o c u l a t i o n Stained With FITC Conjugated Powassan Virus S p e c i f i c Rabbit Gamma Gl o b u l i n 125 Plate 21 S a g i t t a l Section o f Powassan Virus Infected Mouse Brain at the Interfaces Between the Arachnoid and Pi a Mater and the Cerebral Cortex Three Days A f t e r Aerosol I n o c u l a t i o n ; Stained With FITC Conjugated Powassan Virus S p e c i f i c Rabbit Gamma G l o b u l i n 127 Plate 22 S a g i t t a l Section o f Powassan Virus Infected Mouse Brain Showing Widespread Powassan Virus S p e c i f i c Fluorescence i n C e l l s o f the Cerebral Cortex S i x Days A f t e r Aerosol I n o c u l a t i o n 127 Plate 23 E l e c t r o n Micrograph o f Lung T i s s u e Macrophage Three Days A f t e r Intranasal I n s t i l l a t i o n o f Powassan V i r u s ; S t a i n e d with Osmium Tetroxide 129 Pl a t e 24 E l e c t r o n Micrograph o f Nasal;Epithelium S i x Days A f t e r Intranasal I n s t i l l a t i o n o f Powassan V i r u s ; S t a i n e d With Osmium Tetroxide 131 x Page. Pl a t e 25 E l e c t r o n Micrograph o f Nasal E p i t h e l i u m S i x Days A f t e r Intranasal I n s t i l l a t i o n o f Powassan V i r u s ; S t a i n e d With Osmium Tetr o x i d e 133 Plate 26 E l e c t r o n Micrograph o f Nasal E p i t h e l i u m Six Days A f t e r Instranasal I n s t i l l a t i o n o f Powassan Vi rus; Stained With Osmium Tetroxide 135 xi ACKNOWLEDGEMENT The author wishes to express h i s s i n c e r e thanks and a p p r e c i a t i o n t o : Dr. D. M. McLean, P r o f e s s o r and Head o f the D i v i s i o n o f Medical Micro-biology i n the Department o f Mic r o b i o l o g y , U n i v e r s i t y o f B r i t i s h Columbia, s u p e r v i s o r o f the t h e s i s p r o j e c t , f o r h i s guidance and encouragement; Dr. J . J . R. Campbell, P r o f e s s o r and Head of the Department o f Micro-biology and Dr. I. McT. Cowan, Dean of the Faculty of Graduate S t u d i e s , Uni-v e r s i t y o f B r i t i s h Columbia, f o r a l l o w i n g the major p o r t i o n o f t h i s research to be completed at the Defence Research Establishment S u f f i e l d , Ralston, A l b e r t a ; The Defence Research Board f o r s c h o l a r s h i p funds which helped to finance t h i s r esearch; Mr. J . F. C u r r i e , Head o f the Microbiology S e c t i o n a t the Defence Research Establishment S u f f i e l d f o r h i s advice and encouragement; Dr. D. S. Willoughby, formerly o f the Defence Research Establishment S u f f i e l d and Dr. W. C. Stewart, D i r e c t o r o f Research f o r the Defence Research Establishment S u f f i e l d , extramural a d v i s o r s ; My w i f e , Marie, f o r ty p i n g t h i s t h e s i s ; The Instrumentation, Photography and Computer S e c t i o n and the Informa-t i o n S e r v ices Group, Defence Research Establishment S u f f i e l d f o r the prepara-t i o n o f the f i n a l copies o f the t h e s i s . x i i 1 HISTORICAL REVIEW  Introduct i o n Statement o f Purpose Human vi r u s i n f e c t i o n s acquired i n the l a b o r a t o r y , e s p e c i a l l y those due to a r b o v i r u s e s , have been a t t r i b u t e d to the i n h a l a t i o n o f i n f e c t i o u s a e r o s o l s generated during a c c i d e n t s . Arboviruses are main-t a i n e d i n nature p r i n c i p a l l y through b i o l o g i c a l transmission between s u s c e p t i b l e v e r t e b r a t e hosts by blood-sucking arthropods i n whose t i s s u e s they m u l t i p l y . The usual means by which humans c o n t r a c t arbovirus i n f e c -t i o n s i s through b i t e s by i n f e c t e d arthropods, such as t i c k s and mosquitoes. Thus v i r u s i s introduced d i r e c t l y i n t o the blood stream. However, l a b -o r a t o r y i n f e c t i o n s with Venezuelan equine e n c e p h a l i t i s v i r u s (Korpowski and Cox, 1947), St. Louis e n c e p h a l i t i s v i r u s (Magnus, 1950), Russian spring-summer e n c e p h a l i t i s v i r u s (Haymaker e_t aU 1955), West N i l e v i r u s ( N i r , 1959), Kyasanur f o r e s t disease v i r u s (Morse ejt al_, 1962), and Lassa v i r u s (Frame et al_, 1970), ( L e i f e r e_t aj_, 1970), and other arboviruses have r e s u l t e d from i n h a l a t i o n o f v i r u s - l a d e n aerosols f o l l o w i n g l a b o r a t o r y a c c i d e n t s . The lung parenchyma has thereby become the p o r t a l o f entry of v i r u s . Although abundant information i s a v a i l a b l e regarding the patho-genesis of i n f l u e n z a v i r u s which remains l o c a l i z e d to the r e s p i r a t o r y t r a c t , r e l a t i v e l y few i n v e s t i g a t i o n s have been made on the f a t e of i n h a l e d agents such as arboviruses where i n f e c t i o n of vertebrates i s r e g u l a r l y accompanied by viremia. 2 This p r o j e c t reports i n v e s t i g a t i o n s o f the pathogenesis i n mice o f Powassan v i r u s , the only North American member o f the Russian spring-summer complex o f tick-borne group B a r b o v i r u s e s , f o l l o w i n g i n t r a n a s a l i n s t i l l a t i o n o f i n f e c t i o u s d r o p l e t s c o n t a i n i n g a suspen-sio n o f Powassan v i r u s and a f t e r i n h a l a t i o n o f i n f e c t i o u s Powassan viru s a e r o s o l s . This provides a model o f the m u l t i p l i c a t i o n and move-ment of a v i r u s i n man f o l l o w i n g a c c i d e n t a l airborne i n f e c t i o n with an agent which i s n a t u r a l l y arthropod-borne. A necessary p r e r e q u i s i t e to i n f e c t i o n o f mice with suspensions and aerosols o f Powassan v i r u s was the determination o f the decay rate o f v i r u s i n f e c t i v i t y i n suspension and aerosols under various environmental c o n d i t i o n s . Arbovirus Group C l a s s i f i c a t i o n and Pr o p e r t i e s Arboviruses are agents which are maintained i n nature p r i n c i -p a l l y by cy c l e s o f i n f e c t i o n i n v o l v i n g vertebrates and hematophagous arthropods. They induce viremia i n vertebrate h o s t s , at t i t r e s which i n f e c t arthropods which i n g e s t blood. They m u l t i p l y i n the t i s s u e s o f arthropods, and a f t e r an e x t r i n s i c i n c u b a t i o n p e r i o d , they are tr a n s m i t t e d to other vertebrates by b i t e s o f arthropods (Casals and Reeves, 1965). P r o v i s i o n a l c l a s s i f i c a t i o n as an arbovirus i s based on a com-bi n a t i o n o f l a b o r a t o r y and epidemiologic determinants ( C a s a l s , 1961), i n c l u d i n g : circumstances o f i s o l a t i o n , a n t i g e n i c r e l a t i o n s h i p to an e s t a b l i s h e d member, e f f e c t s on l a b o r a t o r y animals and t i s s u e c u l t u r e s , and reduction i n i n f e c t i v i t y t i t r e on exposure to e t h y l ether (Andrewes and Horstmann, 1949), or sodium deoxycholate ( T h e i l e r , 1957). Please see i n s e r t overpage. (Page 2a) (Page 2a) These e c o l o g i c and b i o l o g i c c r i t e r i a are d i f f e r e n t from the p h y s i -c a l and chemical taxonomic c h a r a c t e r i s t i c s used f o r e s t a b l i s h i n g other v i r a l groups (Andrewes, 1962), (Lwoff e_t al_, 1962). There i s no assurance t h a t arbo-viruses w i l l c o n s t i t u t e a homogeneous c l a s s when t h e i r p h y s i c a l and chemical p r o p e r t i e s are more completely s t u d i e d . The number of c u r r e n t l y catalogued arbovirus serotypes has increased d r a m a t i c a l l y from 34 i n December 1949 to 228 i n January 1969 -- American Com-mittee on Arthropod-Borne Viruses (ACAV) (1969); 241 June 1970, ACAV (1970); 269 November 1971, ACAV (1971). Two hundred four arbovirus s t r a i n s as des-c r i b e d i n the Catalogue of Arthropod-Borne Viruses of the World were dispersed among 24 s e r o l o g i c a l groups (ACAV, 1969) (see Table 1). Subsequently new s e r o l o g i c a l groups have been added (ACAV, 1970) (see Table 2). As of November 1971 two f u r t h e r s e r o l o g i c a l groups have been added, namely Matariya and Palyam (ACAV, 1971). Each a n t i g e n i c group includes viruses that c r o s s - r e a c t i n one or more s e r o l o g i c a l t e s t s and thus presumably share common antigens. I t was e a r l i e r proposed that the Tacaribe s e r o l o g i c a l group be includ e d i n a newly defined v i r u s group, the Arenoviruses (Rowe et a]_, 1970). This would e f f e c -t i v e l y remove the Tacaribe complex from the arbovirus group of v i r u s e s . However, ACAV authorized a recommendation to broaden the name of the Catalogue to permit r e t e n t i o n of v i r u s e s p r o v i s i o n a l l y r e g i s t e r e d as arboviruses (such as the Tacaribe group, Lassa v i r u s , e t c . ) . Future e d i t i o n s of the Catalogue w i l l contain (1) arthropod-borne v i r u s e s , (2) p o s s i b l e a r b o v i r u s e s , (3) non-arthropod-borne animal v i r u s e s . The a n t i g e n i c c l a s s i f i c a t i o n and knowledge of i n t e r r e l a t i o n s h i p s have value beyond taxonomic u t i l i t y s i n c e they may r e f l e c t the e v o l u t i o n a r y 3 o r i g i n o f r e l a t e d v i r u s e s and the immune response to these v i r u s e s . An i n f e c t e d host may develop an immune response both to the i n f e c t i n g and r e l a t e d v i r u s e s , but the f i r s t responses tend to be the most s p e c i f i c . A n t i g e n i c i n t e r -r e l a t i o n s h i p s a l s o f a c i l i t a t e the r a p i d i d e n t i f i c a t i o n through grouping o f newly i s o l a t e d v i r u s s t r a i n s ; however they complicate the i n t e r p r e t a t i o n o f r e s u l t s o f s e r o l o g i c a l t e s t s on sera from suspected c l i n i c a l cases or animals i n areas where c l o s e l y r e l a t e d v i r u s e s c o - e x i s t (Casals and Reeves, 1965). Only f o u r f a m i l i e s o f blood sucking arthropods, C u l i c i d a e , Cerato-pogonidae, Psychodidae and Ixodidae are c l e a r l y i m p l i c a t e d as natural vectors of arboviruses ( T a y l o r , 1967). D e t a i l e d f i e l d i n v e s t i g a t i o n and la b o r a t o r y s t u d i e s o f v e c t o r - v i r u s a s s o c i a t i o n s have been l i m i t e d l a r g e l y to vi r u s e s that pose s i g n i f i c a n t p u b l i c health or v e t e r i n a r y problems. Those arboviruses s t u d i e d by e l e c t r o n microscopy e x h i b i t mostly cubic symmetry and are surrounded by envelopes. However some viruses with h e l i c a l or complex symmetry are i n c l u d e d i n the group by a r b o v i r o l o g i s t s (McLean, 1968). Arboviruses contain r i b o n u c l e i c a c i d (RNA) and the majority range i n s i z e between 20 and 60 nanometers (nm.). As p r e v i o u s l y mentioned, arboviruses are transmitted i n nature from one ve r t e b r a t e host to another by the b i t e s o f blood-feeding arthropods e s p e c i a l l y mosquitos and t i c k s . Before transmission can occur, however, s u f f i c i e n t time i s re q u i r e d f o r the v i r u s to m u l t i p l y w i t h i n the t i s s u e s of the arthropod and obtain a s u f f i c i e n t l y high t i t r e i n the s a l i v a r y glands to permit t r a n s f e r o f enough v i r u s to i n f e c t the new host when s a l i v a i s i n t r o -duced during the act o f ta k i n g a second blood meal. In the arthropod the period between o b t a i n i n g an i n f e c t i o u s blood meal and development o f the a b i l i t y to transmit the v i r u s by b i t i n g i s termed the ' e x t r i n s i c incubation p e r i o d 1 . Most arboviruses are transmitted by c u l i c i n e mosquitos, however 4 members of the Russian spring-summer cbmplex o f group B t i c k - b o r n e arbo-v i r u s e s of which Powassan i s a member, are transmitted by b i t e s o f Ixodid t i c k s . Table 3 i s a modified l i s t o f the prototype s t r a i n s of the Russian spring-summer complex o f tick-borne group B a r b o v i r u s e s , y e a r and l o c a t i o n o f i n i t i a l i s o l a t i o n , geographical d i s t r i b u t i o n , t i c k v e c t o r , mammalian r e s e r v o i r and syndromes i n man (McLean, 1968). Because l i t t l e work has been done along these l i n e s , the f o l l o w i n g i s a n e c e s s a r i l y b r i e f account o f the various p h y s i c a l and chemical proper-t i e s of the group B arboviruses. The i n f e c t i v i t y of the group B v i r u s e s i s r e l a t i v e l y s e n s i t i v e to h e a t i n g , i n a c t i v a t i o n o c c u r r i n g at 56° to 60° C f o r 10 to 30 minutes, and i s unstable at room temperature, e s p e c i a l l y at high d i l u t i o n s . I n a c t i v a t i o n can be retarded by the use o f a p r o t e i n d i l u e n t i n s t e a d o f s a l i n e , or the a d d i t i o n of p r o t e i n i n the form o f bovine albumin (0.75 per cent) or animal serum (10 per c e n t ) . The i n f e c t i v i t y and hemagglu-t i n a t i n g a c t i v i t y are best preserved i f crude e x t r a c t s of the v i r u s are l y o -p h i l i z e d or s t o r e d frozen at -70°C. Crude group B arbovirus suspensions are most s t a b l e at pH's ranging between 7.4 to 8.5 with i n f e c t i v i t y being r a p i d l y destroyed at extremely high or low pH. I n f e c t i v i t y of group B arboviruses i s destroyed o r markedly lowered f o l l o w i n g treatment with ethyl ether and acetone, f o r m a l i n , and deoxycholate 1:1000 f o r 30 minutes at 22°C. As p r e v i o u s l y mentioned the l a t t e r i n a c t i -vates a l l arboviruses thus f a r i n v e s t i g a t e d . Members o f the t i c k - b o r n e enceph-a l i t i s complex are i n a c t i v a t e d by e t h a n o l , hydrogen peroxide, t r i c h l o r a c e t i c a c i d , while treatment with fluorocarbons lowers i n f e c t i v i t y only s l i g h t l y . The hemagglutinins of group B arboviruses are most s t a b l e i n b u f f e r e d s a l i n e at pH 9.0 and temperatures near 0°C. They react with erythrocytes o f geese, roosters and newly hatched chicks only i n a narrow pH zone, the optimal 5 pH f o r most members being i n the range 6.0 to 7.0. They are i n a c t i v a t e d by treatment with u l t r a v i o l e t l i g h t while treatment with acetone has l i t t l e e f f e c t on a c t i v i t y . The hemagglutinin can be blocked by various i n h i b i t o r s found i n s e r a and t i s s u e e x t r a c t s (McLean, 1968), (Sokol, 1962), (Hammon and Sather, 1969), (Casals and Brown, 1954). Data on s i z e and morphology of the various members of the group vary depending on the methods o f i n v e s t i g a t i o n i n d i c a t i n g that more work i s needed in t h i s area with p o s s i b l e s t a n d a r d i z a t i o n of methods f o r the various i n v e s t i g a t i o n procedures. E n c e p h a l i t i s caused by i n f e c t i o n with tick-borne group B arboviruses occurs i n e i t h e r s p o r a d i c or epidemic form throughout Europe and A s i a i n areas l o c a t e d between the l a t i t u d e s o f 45°N and 62°N, extending from Scotland i n the west to Kamchatka i n the e a s t . I n f e c t i o n with the Russian spring-summer complex o f viruses may be manifested e i t h e r as a p a r a l y t i c form o f p o l ^ e n c e p h a l o m y e l i t i s or as b i p h a s i c meningoencephalitis. The p a r a l y t i c form r e s u l t s i n f l a c c i d p a r a l y s i s . Bulbar centers may a l s o be a f f e c t e d , causing p a r a l y s i s o f r e s p i r a t i o n , the p a l a t e and the pharynx. In f a t a l cases death u s u a l l y occurs four to seven days a f t e r onset of i l l n e s s . In p a t i e n t s who recover p a r a l y s i s i s f r e q u e n t l y permanent (McLean, 1968). The meningoencephalitis form shows a b i p h a s i c course i n most cases. F i f t y to 70 lymphocytes per cu. mm. appear i n the cerebrospinal f l u i d . The pyramidal, thalamic, c e r e b e l l a r , sympathetic and parasympathetic systems may a l l show d i s o r d e r s , but f l a c c i d p a r a l y s i s s i m i l a r to p o l i o m y e l i t i s does not develop, Complete recovery i s the r u l e but convalescence may be prolonged f o r two months or more (McLean, 1968). 6 Powassan Virus Powassan v i r u s i s a n t i g e n i c a l l y r e l a t e d t o , but d i s t i n c t from Russian spring-summer e n c e p h a l i t i s v i r u s ( C a s a l s , 1960) and i s so f a r the only serotype among the group B tick - b o r n e arboviruses which has been i s o -l a t e d i n North America. The prototype s t r a i n LB was i s o l a t e d from the bra i n of a c h i l d who l i v e d i n a f o r e s t e d area at Powassan, O n t a r i o , Canada ( l a t i -tude 46°N, longitude 79° 30'W) and who had contracted f a t a l e n c e p h a l i t i s (McLean and Donohue, 1959). Subsequent i s o l a t i o n s have been recorded i n Colorado, (Thomas et al_, 1960), South Dakota (McLean et al_, 1964) and New York State (Whitney and Jamnback, 1965), with s e r o l o g i c a l evidence o f i t s presence i n B r i t i s h Columbia (McLean et_ al_, 1968), (McLean et a]_, 1969), (McLean et^ al_, 1971). F i e l d studies have revealed a natural c y c l e i n v o l v i n g rodents such as ground hogs (Marmota monax) and red s q u i r r e l s (Tamiascuris  hudsonicus) as r e s e r v o i r s with hard t i c k s such as Ixodes cookei (Packard), Ixodes marxi (Banks), (McLean et_ al_, 1962), (McLean and Larke, 1963), (McLean et al_, 1964a), (Whitney and Jamnback, 1965), (McLean et al_, 1966), (McLean et a l , 1967), and Dermacentor andersoni ( S t i l e s ) (Thomas e_t al_, 1960), (McLean et a l , 1964a), (McLean e t al_, 1968), (Chernesky, 1969), as ve c t o r s . Man and other vertebrates appear to be t a n g e n t i a l hosts i n the natural t i c k - r o d e n t c y c l e . Abdelwahab ejt al_, (1964) d i s c l o s e d during her i n v e s t i g a t i o n s with Powassan v i r u s i n f e c t i o n o f LLC-MK2 c e l l s t h at the v i r u s produced c y t o p a t h i c e f f e c t s comprising c e l l rounding and cytoplasmic v a c u o l a t i o n f i v e days a f t e r i n o c u l a t i o n , and that mixtures of Powassan v i r u s and s p e c i f i c antiserum i n h i -b i t e d the appearance o f c y t o p a t h i c e f f e c t s . Hemagglutinins f o r r o o s t e r e r t h r o c y t e s f i r s t appeared i n the supernatant f l u i d four days a f t e r i n o c u l a -t i o n , the optimum pH f o r the hemagglutination r e a c t i o n was 6.4 at 22°C. 7 E l e c t r o n microscope s t u d i e s of t h i n s e c t i o n s o f i n f e c t e d t i s s u e c u l t u r e c e l l s revealed p a r t i c l e s 36 to 38 nm. These p a r t i c l e s were u s u a l l y arranged along the outer edges o f c e l l membranes i n a 'palisade' formation or w i t h i n cytoplasmic vacuoles. Negatively s t a i n e d preparations showed i n t a c t v i r u s p a r t i c l e s of 40 to 45 nm. i n diameter. Each p a r t i c l e c o n s i s t e d o f an inner e l e c t r o n dense core of 39 nm. diameter surrounded by a f i n e outer mem-brane of 2 nm. i n width. Newborn mice i n o c u l a t e d with suspensions of Powassan v i r u s developed signs of acute e n c e p h a l i t i s f i v e days a f t e r i n o c u l a t i o n . Weaned mice developed e n c e p h a l i t i s on the seventh day a f t e r i n o c u l a t i o n with the same m a t e r i a l . Pathogenesis and Pathology of Virus Diseases Pathogenesis comprises the sequence of events in m u l t i p l i c a t i o n and movement of v i r u s through the body which may sometimes culminate i n production of disease. Pathology i s concerned with the abnormalities i n t i s s u e s which r e s u l t from the r e a c t i o n to i n j u r y . Viruses m u l t i p l y i n t r a c e l l u l a r ^ and i n the course o f growth a f f e c t the p h y s i o l o g i c s t a t e o f the i n f e c t e d c e l l . The change i s often r e g i s t e r e d as c e l l degeneration or sometimes p r o l i f e r a t i o n . Secondary inflammation u s u a l l y accompanies the primary degenerative or p r o l i f e r a t i v e r e a c t i o n and these com-bined changes o f t e n c o n s t i t u e a pathogenic p i c t u r e c h a r a c t e r i s t i c o f a s p e c i f i c group of v i r u s e s . Pathogenesis may be described i n four d i s t i n c t although c l o s e l y re-l a t e d phases. Phase 1, the entry of the v i r u s from the o u tside i n t o the host, can occur by several routes. The v i r u s can gain entrance to the host by way of the mucus membranes of the nose and t h r o a t , lower r e s p i r a t o r y t r a c t , gastro-i n t e s t i n a l t r a c t , c o n j u n c t i v a l sac or the g e n i t o u r i n a r y t r a c t . The v i r u s may breach the s k i n or subcutaneous and muscular t i s s u e by the b i t e s o f arthropods or v e rtebrates. V e r t i c a l transmission may occur i n cases where a v i r a l i n f e c -8 t i o n i s transmitted from mother to o f f s p r i n g across the p l a c e n t a . The 2nd phase o f pathogenesis i n v o l v e s the spread o f v i r u s from the s i t e s of i n i t i a t i o n to s p e c i f i c t a r g e t t i s s u e . Spread can be media-ted through passage o f the v i r u s from c e l l to c e l l by the formation o f i n t e r -c e l l u l a r b r idges. This type of spread i s r e l a t i v e l y unimportant i n the over-a l l scheme o f v i r a l pathogenesis. One o f the key r o l e s i n the spread o f v i r u s e s from the s i t e o f i n i t i a l i n f e c t i o n must be a s c r i b e d to the macrophages of the r e t i c u l o e n d o t h e l i a l system. Macrophages are s i t u a t e d i n a l l major compartments of the body. Those l i n i n g the l i v e r , spleen and bone marrow s i n u s o i d s monitor the blood and remove f o r i n s t a n c e , c i r c u l a t i n g e f f e t e red blood c e l l s . Those l i n i n g the sinuses o f the lymph nodes monitor the lymph, removing i n e r t p a r t i c l e s or microorganisms brought i n by the a f f e r e n t lymph stream. Those l i n i n g the p l e u r a l and p e r i -toneal c a v i t i e s monitor these c a v i t i e s and those l i n i n g the r e s p i r a t o r y t r a c t monitor the r e s p i r a t o r y f l u i d f i l m i n g e s t i n g i n h a l e d dust p a r t i c l e s and micro-organisms. F i n a l l y , l arge numbers o f macrophage l i v e i n , move through, and monitor the connective t i s s u e spaces throughout the body. The outcome o f v i r u s p e n e t r a t i o n i n t o the host i s to a l a r g e extent determined by the f a t e o f the v i r u s once i t has entered the macrophage. The v i r u s , whether i t i s adsorbed to lymphocytes or taken up by wandering t i s s u e macrophage i s t r a n s -ported by the lymph to the lymph node. The i n f e c t i o n may be overcome i n the nodes through the i n t e r a c t i o n o f antibody and macrophages l i n i n g the sinuses o f the node, but often the v i r u s i s not i n a c t i v a t e d a t the lymph node and i s c a r r i e d by the lymph to the blood stream. One o f the most c h a r a c t e r i s t i c features o f v i r a l i n f e c t i o n s i s the presence of viremia. This means that v i r u s can be i s o l a t e d from the blood o f the i n f e c t e d animal. In the e a r l y stages o f spread w i t h i n the body, 9 viru s e s may ent e r the blood stream a f t e r i n i t i a l m u l t i p l i c a t i o n at the s i t e o f e n t r y ; t h i s i s spoken o f as primary viremia. A secondary viremia may occur l a t e r f o l l o w i n g another stepwise increase o f v i r u s i n the v i s c e r a . Neurotropic viruses can spread along nerve f i b r e s . This has been i n v e s t i g a t e d by t i t r a t i o n o f segments of nerve trunks a t various i n t e r v a l s a f t e r p e r i p h e r a l i n j e c t i o n , by t r a n s e c t i o n experiments and by immunofluores-cence techniques. Phase 3 o f pathogenesis involves the l o c a l i z a t i o n o f v i r u s i n t a r g e t organs. Viruses e x h i b i t a wide range o f v a r i a t i o n i n regard to the organs of the body on which they e x e r t t h e i r main a c t i o n . Each has a s e l e c t i v e a f f i n i t y f o r one o r more such t a r g e t organs and t h i s property i s termed tropism. V i -ruses t h e r e f o r e can be c l a s s i f i e d i n terms o f t h e i r t i s s u e tropism, dermotropic, pneumotropic, n e u r o t r o p i c , e t c . Some vi r u s e s may attack a v a r i e t y o f t i s s u e and thus may be termed p a n t r o p i c . The f i n a l phase of pathogenesis involves the re l e a s e o f v i r u s from the i n f e c t e d host. Viruses may be relea s e d from a v a r i e t y o f s i t e s ; the s i t e o f l o c a l m u l t i p l i c a t i o n , the upper o r lower r e s p i r a t o r y t r a c t , the g a s t r o i n -t e s t i n a l or g e n i t o u r i n a r y t r a c t . Virus may a l s o be relea s e d i n the blood --an event which i s of p a r t i c u l a r i n t e r e s t when one considers the t r a n s f u s i o n of whole blood o r when one considers the arthropod-borne group of vi r u s e s (Bang and L u t t r e l l , 1961), (Buddingh, 1965), (Rhodes and D i t c h f i e l d , 1968), (Mims, 1964). V i r a l Invasion o f the Central Nervous System The c l i n i c a l m anifestations o f many v i r u s i n f e c t i o n s are dependent on whether or not the v i r u s gains access to s u s c e p t i b l e c e l l s w i t h i n the cen t r a l nervous system. I f i n f e c t i o n i s l i m i t e d to extraneural t i s s u e , signs may be mi l d o r inapparent, but i n f e c t i o n of neural t i s s u e s may lead to menin-g i t i s , e n c e p h a l i t i s , p a r a l y s i s , and death. T h e r e f o r e , the mechanisms by 10 which vi r u s e s penetrate the c e n t r a l nervous system are o f prime importance i n the understanding o f the pathogenesis o f v i r u s diseases which have nervous system complications. I n f e c t i o n o f the c e n t r a l nervous system with viruses can no longer be explained on the basis o f rare agents with a s p e c i a l p r e d i l e c t i o n f o r neural t i s s u e s . E n t e r o v i r u s e s , mumps v i r u s , herpes v i r u s , measles v i r u s , and arthropod-borne vi r u s e s are the agents most o f t e n r e s p o n s i b l e f o r i n f e c t i o n s i n v o l v i n g the c e n t r a l nervous system. S e r o l o g i c a l evidence supports the f a c t s that large segments of the population have been i n f e c t e d with v i r a l agents but have not dis p l a y e d any c l i n i c a l signs o f disease. P o l i o v i r u s e s and the arthropod-borne e n c e p h a l i t i s v i r u s e s cause c l i n i c a l l y s i g n i f i c a n t diseases on r e l a t i v e l y rare occasions. These diseases are fr e q u e n t l y manifest by neural involvement. Herpes, mumps and measles i n f e c t i o n s u s u a l l y present as mild i l l n e s s e s , but may sometimes be complicated by c e n t r a l nervous system involvement (Meyer e_t a l , 1960). E a r l y development o f antibody, o r a d m i n i s t r a t i o n o f antiserum at the time o f extensive v i r a l r e p l i c a t i o n i n the c e n t r a l nervous system may pro-mote extensive neuronal damage and death o f mice i n f e c t e d with Langat v i r u s (Webb, et al_, 1968), (Webb et-al . , 1968a). In human i n f e c t i o n s i t i s not p o s s i b l e to t r a c e the pathogenesis o f e n c e p h a l i t i c diseases caused by v i r u s e s , and i n the case o f f a t a l i t i e s caused by such i n f e c t i o n s , the disease i s u s u a l l y too widespread to re c o n s t r u c t i t s e v o l u t i o n . Attempts have been made to study the t i s s u e changes due to v i r u s i n f e c t i o n s i n experimental animals. This involves the examination o f t i s s u e c o l l e c t e d at various times during the incubation p e r i o d . Conventional h i s t o -l o g i c a l examination o f t i s s u e i s g e n e r a l l y inadequate because morphological changes due to v i r a l i n f e c t i o n s occur l a t e i n the course o f i n f e c t i o n , i f they occur at a l l . 11 Fenner (1949) i n h i s studies o f e c t r o m e l i a v i r u s i n f e c t i o n s i n mice (mousepox) used a more p r e c i s e method to study the pathogenesis o f the disease. Animals were d i s s e c t e d at r e g u l a r i n t e r v a l s during the i n c u b a t i o n p e r i o d and organs were t i t r a t e d to q u a n t i t a t e v i r u s content. This method demonstrates the rate o f v i r a l m u l t i p l i c a t i o n and the sequence o f i n f e c t i o n o f the various organs. However, i t does not i d e n t i f y the c e l l s w i t h i n the t i s s u e which are a f f e c t e d and i n cases where there i s a pronounced vir e m i a , does not d i s t i n g u i s h between the blood-borne v i r u s and the v i r u s produced in s i t u . The development of the f l u o r e s c e n t - a n t i b o d y technique (Coons et a l , 1942), (Coons et_ al_, 1950), provided a p r a c t i c a l means f o r t r a c i n g v i r a l i n f e c t i o n at the c e l l u l a r l e v e l . The technique e s s e n t i a l l y i n v o l v e s the pro-duction o f a hyperimmune serum against the v i r u s causing the i n f e c t i o n ; con-j u g a t i o n o f the i s o l a t e d hyperimmune gamma g l o b u l i n f r a c t i o n with a f l u o r e s -cent l a b e l , and a p p l i c a t i o n o f the l a b e l l e d g l o b u l i n to s e c t i o n s of t i s s u e . The l a b e l l e d g l o b u l i n complexes with viruses i n i n f e c t e d t i s s u e r e v e a l i n g f o c i o f s p e c i f i c f l uorescence when examined using a microscope with an u l t r a -v i o l e t l i g h t source. A combination of the f l u o r e s c e n t - a n t i b o d y technique together with the q u a n t i t a t i o n of v i r u s i n various t i s s u e s provide p r e c i s e methods f o r the accurate p o r t r a y a l of the pathogenesis o f a v i r u s disease (Mims and Subrahmanyan, 1966). Neural Spread of Virus to the Central Nervous System Observations have been made th a t the c e n t r a l nervous system repre-sents a complicated meshwork o f t i g h t l y packed c e l l s and processes with v i r -t u a l l y no f r e e space with the exception o f minute c l e f t s of 100 - 200 Angstrom units between adjacent elements (Shcale and Ford, 1965). Presumably these tortuous c l e f t s between neural elements would preclude the d i s p e r s i o n of v i -ruses by way of the i n t e r c e l l u l a r spaces o f the c e n t r a l nervous system. How-12 ever, Bodian (1948) studying the development of morphological l e s i o n s i n experimental p o l i o m y e l i t i s and L u t t r e l l and Bang (1958) with s i m i l a r s t u d i e s using Newcastle disease v i r u s obtained evidence which i n d i c a t e d that the pathway o f spread was neuronal, although i t was not decided whether the spread was by way o f the axon or i n t e r c e l l u l a r space. Boyse e t al^ (1956a), studying the spread of herpes simplex i n the s p i n a l cord of r a b b i t s and L i u et al_ (1958) studying the p o l i o v i r u s i n f e c t i o n s i n monkeys concluded that the spread was from c e l l to c e l l through the i n t e r c e l l u l a r space. I t was f u r t h e r suggested by Boyse e t al_ (1956) that an important f a c t o r supporting the d i s p e r s a l o f viruses or b a c t e r i a l toxins through the cerebrospinal axis may be the repeated f l u c t u a t i o n o f pressure conveyed onto the c e n t r a l nervous system by the impact of the a r t e r i a l pulse. In strong support o f the theory o f v i r a l d i s p e r s i o n through the e x t r a c e l l u l a r space are the observations that high doses o f hyperimmune serum can l i m i t or a r r e s t the progress o f an i n f e c t i v e e n c e p h a l i t i c process ( L i u e_t al_, 1959), ( S c h i n d l e r , 1961). It was long b e l i e v e d that the normal hematoencephalic b a r r i e r was impermeable to most i f not a l l v i r u s e s (Hurst, 1936). E a r l y experimental s t u d i e s with rabies and herpes simplex v i r u s e s suggested that v i r u s e s spread to the c e n t r a l nervous system along p e r i p h e r a l nerves (Marinesco and Dragenesco 1923), (Goodpasture and l e a g u e , 1923), (Goodpasture, 1925). The neural lymphatics have a l s o been i m p l i c a t e d as p o t e n t i a l path-ways f o r the spread of v i r u s to the c e n t r a l nervous system, but anatomical studies conducted by B r i e r l e y (1950) have i n d i c a t e d that they l i e o u t s i d e the perineurium and do not communicate d i r e c t l y with the subarachnoid space. The subarachnoid space around nerve roots terminates i n a cul-de-sac near the proximal pole o f the ganglion. F i e l d and B r i e r l e y (1948) (1948a) examined the d i f f u s i o n o f p a r t i c l e s from India ink w i t h i n the subarachnoid space and 13 found t h a t they d i f f u s e through the c u f f o f arachnoid surrounding the p e r i -pheral nerves and are taken up by the pr e v e r t e b r a l lymphatics. The d i f f u s i o n i s normally c e n t r i f u g a l , but when abdominal pressure was a p p l i e d , r e v e r s i n g the pressure g r a d i e n t , p a r t i c l e s were forc e d from the lymphatics i n t o the dorsal root g a n g l i a . In a review of nerve trunks as pathways of i n f e c t i o n Wright (1953) concluded that three conduits were a v a i l a b l e : axons, lymphatics and i n t e r -c e l l u l a r space o f nerve f a s i c l e s . Axons were e l i m i n a t e d as a pathway because of the v i s c o s i t y o f axoplasm, the lymphatics because of the c e n t r i f u g a l flow of lymph (except under i r r e g u l a r c o n d i t i o n s ) thus l e a v i n g t i s s u e spaces between i n d i v i d u a l nerve f i b r e s as the l o g i c a l pathway f o r the d i s p e r s i o n o f v i r u s e s . Radiopaque substances o r dyes i n j e c t e d d i r e c t l y i n t o a nerve f a s i c l e spread q u i c k l y toward the subdural space (Dorang and Matzke, 1960) without l e a k i n g v i s i b l y i n t o the epineurium. The perineurium therefore seems to be a r a t h e r t i g h t semipermeable membrane. The d i s p e r s i o n occurs i n t i s s u e spaces o f the endoneurium. The d r i v i n g f o r c e o f f l u i d w i t h i n the f a s i c l e s i s b e l i e v e d to a r i s e from the pressure developed during normal p h y s i o l o g i c a l a c t i v i t y o f the muscle (Wright, 1959). Rabies has been quoted as the c l a s s i c a l example o f a v i r u s disease which spreads to the ce n t r a l nervous system along p e r i p h e r a l nerves (Habel, 1964). I t has been observed t h a t there was a r a p i d d i s p e r s i o n o f the v i r u s to the cen t r a l nervous system i n s p i t e o f no (Johnson, 1965a), or l i m i t e d (Schind-l e r , 1961) r e p l i c a t i o n at the s i t e o f i n o c u l a t i o n . A f t e r m u l t i p l i c a t i o n w i t h i n the c e n t r a l nervous system the v i r u s extends c e n t r i f u g a l l y along p e r i p h e r a l nerves (Dean e_t al_, 1963). In Johnson's (1965) study using f i x e d - r a b i e s v i r u s i n mice, no e v i -dence o f endon e u r a l - c e l l i n f e c t i o n was found. Spread o f v i r u s to the c e n t r a l nervous system appeared to be by way of the nerve s i n c e i n f e c t i o n could be 1 4 prevented by t r a n s e c t i n g the appropriate p e r i p h e r a l nerve. When the nerve was maintained i n t a c t , the appropriate dorsal route ganglion c e l l s were f i r s t to show evidence o f i n f e c t i o n upon fl u o r e s c e n t - a n t i b o d y s t a i n i n g . Johnson (1964) using r o u t i n e t i t r a t i o n procedures and f l u o r e s c e n t -antibody s t a i n i n g f o r the i d e n t i f i c a t i o n of i n f e c t e d c e l l s showed that a f t e r i i n t e r c e r e b r a l i n o c u l a t i o n of herpes simplex v i r u s , the v i r u s r a p i d l y d i s -persed i n t o the cerebrospinal f l u i d , m u l t i p l i e d i n the meninges and ependyma and then invaded the underlying parenchyma o f the b r a i n i n f e c t i n g both neurons and g l i a . Following extraneural i n o c u l a t i o n he found that v i r u s gained access to the c e n t r a l nervous system by hematogenous and neural path-ways. A f t e r i n t r a p e r i t o n e a l and i n t r a n a s a l i n o c u l a t i o n , v i r u s was found to m u l t i p l y i n v i s c e r a and produce a viremia; f o c i o f c e n t r a l nervous system i n f e c t i o n then developed around cere b r a l v e s s e l s . Spread of v i r u s was demon-s t r a t e d along p e r i p h e r a l nerves a f t e r subcutaneous and i n t r a n a s a l i n o c u l a t i o n . Johnson s t a t e s t h a t t h i s spread r e s u l t e d from c e n t r i p e t a l i n f e c t i o n o f endo-neural c e l l s (Schwann c e l l s and f i b r o b l a s t s ) and a l s o t h a t v i r a l antigen was not found i n axons even a f t e r i n f e c t i o n of the corresponding ganglion c e l l p e r i karyon. I t would appear th e r e f o r e that i n f e c t i o n of endoneural c e l l s would provide a pathway f o r the spread of v i r u s along nerves together with the a l t e r n a t i v e o f spread w i t h i n the i n t e r s p a c e s o f nerve f a s i c l e s ( F i e l d , 1952), (Dean et al_, 1963), (Wildy, 1967). There are several other v i r u s e s which have been shown to be capable of i n f e c t i n g and progressing along p e r i p h e r a l nerves: human p o l i o v i r u s (Nathanson and Bodian, 1961), West N i l e v i r u s (Kundin e t al_, 1962), t i c k -borne e n c e p h a l i t i s v i r u s ( A l b r e c h t , 1962). Studies by Johnson with herpes simplex i n f e c t i o n i n mice showed t h a t a f t e r i n t r a n a s a l i n o c u l a t i o n v i r u s could spread to the c e n t r a l nervous system by neural and hematogenous route simultaneously (Johnson, 1964). 15 Hematogenous Spread o f Virus to the Central Nervous System As p r e v i o u s l y mentioned the 'blood-brain b a r r i e r ' was considered to be impermeable to vi r u s e s (Hurst, 1936). Extensive studies on the patho-genesis o f p o l i o m y e l i t i s i n the 1940 1s and 1950's (Bodian, 1955), (Sabin, 1956), (Bodian and Horstmann, 1965) provided the major impetus f o r the recon-s i d e r a t i o n o f the hematogenous spread o f vi r u s e s to the c e n t r a l nervous sys-tem. The hematogenous route f o r the dissemination o f vi r u s e s i s o f para-mount importance i n the pathogenesis o f diseases caused by members o f the arthropod-borne group of v i r u s e s . However, the wide range o f hosts together with the v a r i a t i o n s i n p a t h o g e n i c i t y o f the multitude o f vi r u s e s which com-pose the arbovirus group makes tenuous g e n e r a l i z a t i o n s on pathogenesis with respect to the various d i s e a s e s ; t h e r e f o r e t h i s o u t l i n e w i l l be confined to some o f the arboviruses which are known to cause c e n t r a l nervous system i n f e c -t i o n s i n man and animals such as tick-borne e n c e p h a l i t i s v i r u s e s , o f which Powassan v i r u s i s a member. Most arboviruses have a broad range o f s u s c e p t i b l e animals. How-ever some species are r e s i s t a n t to v i r u s mul t i p ! i c a t i on (Casals and Clarke, 1965), (Clarke and C a s a l s , 1965), (McLean, 1968). Some species can become i n f e c t e d with a v i r u s and show evidence o f widespread systemic involvement with n e g l i g i b l e neural involvement. This type o f v i r u s - h o s t r e l a t i o n s h i p i s found p r i m a r i l y i n the natural vertebrate host animal and probably repre-sents natural s e l e c t i o n which has taken place over long periods o f c l o s e a s s o c i a t i o n between the host and v i r u s (Fenner, 1965). I t was learned that the Princeton R o c k e f e l l e r I n s t i t u t e (PRI) s t r a i n o f mice was h i g h l y r e s i s -t a nt to a l l members o f Casals' group B a r b o v i r u s e s , but not to the group A arbo v i r u s e s . The f a c t o r c o n t r o l l i n g t h i s r e s i s t a n c e behaved i n cros s -breeding experiments as a s i n g l e dominant autosomal gene, which depressed 16 the m u l t i p l i c a t i o n o f the v i r u s i n the brains of r e s i s t a n t mice. I t was concluded that the r e s i s t a n c e was due to the i n a b i l i t y of the v i r u s e s from the group B arboviruses to i n f e c t the macrophages o f the r e s i s t a n t mice (Sabin, 1952), (Goodman and Koprowski, 1962). Thus i t would appear that in the case o f the above experiments r e s i s t a n c e to v i r u s disease may be under genetic c o n t r o l which operates by the a b i l i t y of v i r u s e s to i n f e c t and destroy c e r t a i n key c e l l s . Mims (1964) i n h i s e x c e l l e n t review on the pathogenesis of v i r u s diseases provides information on the clearance of v i r u s e s from the blood by the macrophages o f the r e t i c u l o e n d o t h e l i a l system. He s t a t e s that p a r t i c l e s i z e a f f e c t s the clearance r a t e . Large v i r u s e s such as v a c c i n i a or v e s i c u l a r s t o m a t i t i s are c l e a r e d much more r a p i d l y than the smaller v i r u s e s . He men-tion e d that another c h a r a c t e r i s t i c feature noted with respect to v i r u s c l e a r -ance curves i s that clearance does not continue at a constant r a t e , hence the curve f l a t t e n s out l e a v i n g an 1 uncleared t a i 1 . 1 He suggests that t h i s un-c l e a r e d t a i l may be due to the f a c t that some vir u s e s become a s s o c i a t e d with the c e l l u l a r elements o f the blood stream or that the uptake o f p a r t i c l e s by the r e t i c u l o e n d o t h e l i a l system macrophages i s p a r t l y r e v e r s i b l e with an e q u i -l i b r i u m being reached between c i r c u l a t i n g and phagocytosed p a r t i c l e s . In most v i r u s i n f e c t i o n s , organs and t i s s u e s are i n v o l v e d which are d i s t a n t from the s i t e o f i n i t i a l entry o f the v i r u s i n t o the body and spread to these organs takes place v i a the blood stream. In the case of arbovirus diseases adequate viremia i s e s s e n t i a l f o r the t r a n s f e r o f v i r u s to new hosts by blood feeding arthropods. V i r u s e s , as mentioned e a r l i e r , may become asso-c i a t e d with the formed elements o f the blood stream c e l l s such as thrombo-c y t e s , lymphocytes, monocytes and leukocytes. One important aspect o f white c e l l a s s o c i a t e d viremias i s that leukocytes are able to c a r r y v i r u s e s with them i n t h e i r migration through the body. In some viremias v i r u s becomes 17 a s s o c i a t e d with the e r y t h r o c y t e . R i f t V a l l e y f e v e r v i r u s i s an example of t h i s type o f a s s o c i a t i o n (Mims, 1956). Viruses may als o be re l e a s e d f r e e i n t o the f l u i d o r plasma p o r t i o n o f the blood. This type of plasma viremia i s s u b j e c t to clearance by the r e t i c u l o e n d o t h e l i a l system. I f the rate o f entry o f v i r u s i n t o the blood stream equals the rate o f clearance then the l e v e l o f viremia remains constant. I f the clearance rate i s r a p i d i n re-l a t i o n to the rate o f entry there may be no detectable v i r e m i a . Obviously a r i s e o r f a l l i n the ra t e o f entry o f v i r u s i n t o the blood would be accom-panied by a r i s e o r f a l l i n the degree o f viremia. Many arbovirus i n f e c t i o n s d i s p l a y the above f e a t u r e s . Some viruses are known f o r t h e i r a b i l i t y to i n f e c t and damage v a s c u l a r endothelium. A search f o r a c l o s e a s s o c i a t i o n between v a s c u l a r endothelium and arboviruses has been pursued i n order to e x p l a i n the high l e v e l s o f viremia caused i n arbovirus i n f e c t i o n s ; a l s o i f arboviruses had the a b i l i t y to grow through v a s c u l a r endothelium the pathway of i n v a s i o n o f the c e n t r a l nervous system by various members of the arbo-v i r u s e s could more e a s i l y be understood. Fluorescent-antibody s t u d i e s by various researchers reveal evidence o f v i r a l antigen i n v a s c u l a r endothelium i n the cases o f some arbovirus i n f e c t i o n s , e.g., Sindbis v i r u s (Johnson, 1965); together with lack of a n t i -gen i n v a s c u l a r endothelium i n the cases o f o t h e r s , e.g., tick-borne encepha-l i t i s v i r u s i n mice A l b r e c h t (1962) i n d i c a t e d that viremia i n v a r i a b l y follows subcutaneous i n o c u l a t i o n o f v i r u s . He s t a t e s that the o r i g i n o f the viremia i s obscure. Some authors b e l i e v e t h a t m u l t i p l i c a t i o n o f the v i r u s occurs i n the r e t i c u l o e n d o t h e l i a l system, others were only able to demonstrate that v i r u s proceeded q u i c k l y to the c e n t r a l nervous system with subsequent pouring o f v i r u s i n t o the blood c i r c u l a t i o n . S t i l l other authors were able to demon-s t r a t e that o r a l a d m i n i s t r a t i o n o f v i r u s caused i n f e c t i o n o f the alimentary canal with subsequent spread to the c e n t r a l nervous system and no involvement 18 o f the r e t i c u l o e n d o t h e l i a l system. The m u l t i p l i c i t y o f r e s u l t s obtained were explained on the basis o f d i f f e r e n c e s i n the concentration o f v i r u s i n d i f f e r e n t t i s s u e ; these v a r i a t i o n s being due to i n a c t i v a t i o n of v i r u s by t i s s u e enzymes during processing and t i t r a t i o n or the presence of various volumes o f viremic blood present i n the d i f f e r e n t organs at the time o f pro-c e s s i n g and t i t r a t i o n . However, examination o f t i s s u e using the f l u o r e s c e n t -antibody technique revealed s p e c i f i c fluorescence i n p r a c t i c a l l y a l l neuronal c e l l s i n the b r a i n , medulla and s p i n a l cord a f t e r subcutaneous o r o r a l i n f e c -t i o n of s u c k l i n g mice with tick-borne e n c e p h a l i t i s v i r u s . Furthermore, s p e c i f i c f l uorescence was observed i n the 'ground substance 1 p o i n t i n g to continuous r e l e a s e o f v i r u s from nerve c e l l s with involvement o f g l i a l c e l l s s i n c e b r i g h t rings o f fluorescence were observed around many g l i a l n u c l e i . The propagation and dissemination o f v i r u s along nerve t r a c t s could be well demonstrated i n the o p t i c nerve. The most i n t e n s i v e fluorescence was ob-served i n the p e r i p h e r a l nervous system comprising a l l the sympathetic and parasympathetic g a n g l i a . The a f f i n i t y o f the v i r u s f o r non-nervous t i s s u e was a l s o noted e s p e c i a l l y i n the s e c r e t o r y glands where nests of f l u o r e s c e n t c e l l s were observed i n the p a r o t i d s , naso-lacrymal glands, pancreas, i n small groups of c e l l s i n the g a s t r i c and p y l o r i c mucosa and c o l o n , Kidneys and various endocrine glands a l s o showed s p e c i f i c f l u o r e s c e n c e . In the spleen, lymph nodes and bone marrow s i n g l e or c l u s t e r s o f hemopoietic c e l l s d i s -played ring-shaped f l u o r e s c e n c e . Smooth and s t r i a t e d muscle was also shown to be i n v o l v e d . It was found that i n men autopsied a f t e r f a t a l i n f e c t i o n s due to Russian spring-summer e n c e p h a l i t i s v i r u s involvement o f the p e r i p h e r a l sym-p a t h e t i c and parasympathetic nervous system was usual. Further s t u d i e s on the pathogenesis o f t i c k - b o r n e e n c e p h a l i t i s v i r u s i n mice by Malkova (1962) and Malkova and Smetana (1966) revealed that 1 9 when ti c k - b o r n e e n c e p h a l i t i s v i r u s was administered subcutaneously the v i r u s was transported i n t o the regional lymph vesse l s and then v i a these, penetrated the blood stream. A d i r e c t t r a n s l o c a t i o n of the v i r u s i n t o the blood stream was never observed. I t was found that tick-borne e n c e p h a l i t i s v i r u s spread f i r s t to the lymphatics subsequently c i r c u l a t e d i n the lymph and blood and then invaded other organs i n c l u d i n g the c e n t r a l nervous system. When peak t i t r e s were observed i n the b r a i n most other organs and t i s s u e s were observed to contain v i r u s . In r a b b i t s , however, which are r e s i s t a n t even to i n t r a c e r e b r a l inocu-l a t i o n with tick-borne e n c e p h a l i t i s v i r u s , i n f e c t i o n was l i m i t e d to the lymph-a t i c and blood c i r c u l a t o r y systems. In Czechoslavakia a high incidence o f human i n f e c t i o n with t i c k -borne e n c e p h a l i t i s v i r u s was observed where some 600 people became ill due to the contamination of cows' milk with several l i t r e s of tick-borne encepha-l i t i s v i r u s i n f e c t e d goats' milk. Pogodina (1962) revealed that tick-borne e n c e p h a l i t i s v i r u s administered o r a l l y to mice r e s u l t e d i n a p a r a l y t i c i n f e c -t i o n . The pathogenetic pathway o u t l i n e d was p e n e t r a t i o n of v i r u s i n t o the t i s s u e s o f a l l parts o f the g a s t r o i n t e s t i n a l t r a c t , followed by m u l t i p l i c a -t i o n mainly i n the i n t e s t i n e with subsequent viremia and i n v a s i o n o f the c e n t r a l nervous system. A v a r i e t y of h i s t o l o g i c a l and f l u o r e s c e n t - a n t i b o d y s t u d i e s i n d i -cate that i n f e c t i o n o f the c e n t r a l nervous system occurs by the t r a n s l o c a -t i o n of v i r u s across the blood-brain b a r r i e r at a v a r i e t y of l o c a t i o n s con-cu r r e n t with viremia and that v i r u s enters the b r a i n e a r l y i n the course of the i n f e c t i o n during the r i s e of viremia followed by d i f f u s e e n c e p h a l i t i s in two to three days depending on the arbovirus concerned (Kundin e t al_, 1962), ( A l b r e c h t , 1962), (Johnson, 1965). 2 0 I t appears that a c e r t a i n l e v e l o f viremia i s r e q u i r e d before v i r u s can cross the blood-brain b a r r i e r . In the case of the Peking s t r a i n of Japanese B e n c e p h a l i t i s v i r u s i n f e c t i o n i n mice, viremias i n the region of 1 8 l o g 1 0 L D 5 Q / 0 . 0 3 ml. were requ i r e d to cause e n c e p h a l i t i s (Huang and Wong, 1 9 6 3 ) . A comparison of the p r o l i f e r a t i o n c a p a c i t y of West N i l e v i r u s , t i c k -borne e n c e p h a l i t i s v i r u s (Hypr), Venezuelan equine e n c e p h a l i t i s v i r u s and Japanese B e n c e p h a l i t i s v i r u s (high and low neuroinvasive s t r a i n ) i n p e r i -pheral and neural t i s s u e seems to i n d i c a t e that neuroinvasiveness i s i n v a r i a b l y l i n k e d to the a b i l i t y o f the neuroinvasive v i r u s to m u l t i p l y i n the p e r i p h e r a l t i s s u e which r e s u l t s i n high l e v e l s of viremia ( A l b r e c h t , 1 9 6 8 ) . O l f a c t o r y Spread of Virus to the Central Nervous System The p o s s i b l e mechanisms of spread o f v i r u s e s across the nasal mu-cosa, submucosal t i s s u e and c r i b r i f o r m p l a t e are s i m i l a r to those discussed f o r neural spread, however several s i g n i f i c a n t anatomical d i f f e r e n c e s e x i s t . As with the s p i n a l r o o t , a c u f f o f arachnoid surrounding the o l f a c t o r y - n e r v e f i b r e p e r f o r a t e s the c r i b r i f o r m p l a t e and dura and forms a cul-de-sac i n the submucosal connective t i s s u e . Carbon p a r t i c l e s o r l a b e l l e d p r o t e i n s can d i f f u s e through t h i s c u f f and enter the lymphatic channels i n the o l f a c t o r y submucosa ( B r i e r l e y , 1 9 5 0 ) , ( F i e l d and B r i e r l e y , 1 9 4 8 ) . The normal d i f f u s i o n of cerebrospinal f l u i d across the o l f a c t o r y area has been shown to be ten times greater than the d i f f u s i o n o f cerebrospinal f l u i d from s p i n a l nerve roots i n p r e v e r t e b r a l lymphatics (C o u r t i c e and Simmonds, 1 9 5 1 ) . E l e c t r o n microscopy o f i n f a n t r a b b i t s has shown that o l f a c t o r y receptors extend i n t o and beyond the o l f a c t o r y e p i t h e l i u m , t h e r e f o r e a very d i r e c t communication o f nerve f i b r e s with external environment e x i s t s i n the o l f a c t o r y mucosa. Dyes dropped i n t o the nasal c a v i t i e s o f r a b b i t s have been found to reach the o l f a c t o r y bulbs i n one hour presumably by way o f i n t e r s t i t i a l spaces o f the 21 p e r i n e u r a l sheath o f these o l f a c t o r y nerves (de Lorenzo, I960). Fluorescent-antibody studies i n mice have confirmed the o l f a c t o r y spread o f v i r u s e s to the c e n t r a l nervous system. In Johnson's (1964) study of herpes simplex i n f e c t i o n i n mice by i n t r a n a s a l i n s t i l l a t i o n the p a t t e r n of f l u o r e s c e n t c e l l s showed two types of spread i n t o the o l f a c t o r y bulbs. I n f e c t i o n extended d i r e c t l y from nasal mucosa and submucosal t i s s u e through the meninges i n t o the subarchnoid space; t h i s gave r i s e to widespread i n f e c -t i o n o f meninges, s i m i l a r to that seen a f t e r i n t r a c e r e b r a l i n o c u l a t i o n . In other mice however, i n f e c t i o n o f the o l f a c t o r y bulb was observed with no involvement o f the meninges; f i n e l i n e s o f fluorescence were found t r a v e r -s i n g the c r i b r i f o r m p l a t e and meninges i n a s s o c i a t i o n with o l f a c t o r y nerve f i b r e s . Aerosol i n f e c t i o n of mice with West N i l e v i r u s , a group B arbo-v i r u s , showed no i n f e c t i o n of the nasal mucosa by immunofluorescent tech-niques y e t i n i t i a l i n f e c t i o n of the o l f a c t o r y bulbs occurred. This suggests that v i r u s may spread along o l f a c t o r y f i b r e s without i n f e c t i o n o f submucosal perin e u r a l and endoneural c e l l s ( N i r e t a l _ , 1965). Airborne Virus I n f e c t i o n s Extensive surveys have been published on l a b o r a t o r y i n f e c t i o n s l i s t i n g e t i o l o g i c a l agents, frequency rates f o r d i f f e r e n t s c i e n t i f i c i n s t i -t u t i o n s , the usual l a b o r a t o r y techniques i n v o l v e d i n causing i n f e c t i o n s and methods o f c o n t r o l . ( S u l k i n and Pike, 1951), ( S u l k i n and P i k e , 1951a), (Chatigny, 1961), ( P h i l l i p s , 1965). A l a r g e p r o p o r t i o n of these i n f e c t i o n s are due to the i n h a l a t i o n o f microorganisms due to a c c i d e n t a l c r e a t i o n o f i n f e c t i o u s a e r o s o l s . A m u c o c i l i a r y blanket covers much o f the surface of the r e s p i r a t o r y t r a c t . Airborne p a r t i c l e s are deposited on the mucoid su r f a c e and c i l i a t e d e p i t h e l i u m c a r r y the entrapped p a r t i c l e s upward to the trachea and oropharynx. 22 D i s t a l to the r e s p i r a t o r y bronchioles the m u c o c i l i a r y blanket i s absent and macrophages are i m p l i c a t e d as p l a y i n g an important r o l e i n the uptake of inh a l e d f o r e i g n p a r t i c l e s i n t h i s area, c a r r y i n g antigen to the pulmonary lymph nodes (Mims, 1964), (Danes et a l , 1969), (Benda and Danes, 1969). Cer-t a i n v i r u s e s ( e n t e r o v i r u s e s , tick-borne e n c e p h a l i t i s v i r u s ) c a r r i e d upward by the m u c o c i l i a r y blanket may subsequently i n f e c t the g a s t r o i n t e s t i n a l t r a c t a f t e r swallowing. Penetration i n t o and r e t e n t i o n o f airborne v i r u s e s i n the lung i s dependent on the s i z e o f the i n h a l e d p a r t i c l e . Experiments conducted along t h i s l i n e r e p ort that p e n e t r a t i o n i n t o the pulmonary a i r spaces i s nearly zero f o r p a r t i c l e s of 10 microns i n diameter but reaches a maximum at and below 1 micron and als o that the percentage d e p o s i t i o n o f p a r t i c l e s which have penetrated to the a l v e o l a r a i r spaces i s maximum f o r p a r t i c l e s between 1 and 2 microns. I t was noted, however that the p r o b a b i l i t y f o r d e p o s i t i o n of p a r t i c l e s 1 micron and l e s s decreases u n t i l p a r t i c l e s i z e s o f 0.5 to 0.25 microns are reached when the p r o b a b i l i t y f o r d e p o s i t i o n again r i s e s . I t was pointed out that those p a r t i c l e s i n the 1 to 2 micron range which do reach the a l v e o l i s e t t l e out by g r a v i t y whereas p a r t i c l e s 0.5 microns and l e s s are p r e c i p i t a t e d by forces of d i f f u s i o n , the d i f f u s i o n forces i n c r e a s i n g as the p a r t i c l e s i z e f u r t h e r diminishes (Hatch 1961), (Hatch and Gross, 1964). The majority o f the l a b o r a t o r y i n f e c t i o n s acquired by workers engaged i n research with arboviruses appears to be due to the acc i d e n t a l c r e a t i o n o f i n f e c t i o u s a e r o s o l s . The types o f accidents l e a d i n g to the pro-duction o f i n f e c t i o u s aerosols are: c a r e l e s s use of o r handling o f p i p e t t e s , homogenizers, c e n t r i f u g e s , syringes and excretions from i n f e c t e d animals. Laboratory i n f e c t i o n s caused by the f o l l o w i n g arboviruses have been reported: Venezuelan equine e n c e p h a l i t i s v i r u s (Koprowski and Cox, 1947), St. Louis e n c e p h a l i t i s v i r u s (Magnus, 1950), Russian spring-summer e n c e p h a l i t i s v i r u s 23 (Haymaker e_t aj_, 1955), West N i l e v i r u s ( N i r , 1959), and Kyasanur f o r e s t disease v i r u s (Morse ejt al_, 1962), Lassa v i r u s (Frame et al_, 1970), ( L e i f e r e t al_, 1970). The study o f the pathogenesis of r e s p i r a t o r y i n f e c t i o n s caused by viruses by the experimental i n o c u l a t i o n o f l a b o r a t o r y animals with aerosols of i n f e c t i o u s v i r u s e s has provided information with regard to dose-response r e l a t i o n s h i p s , modes o f spread o f v i r u s e s , v i r e m i a , t a r g e t organs involved and subsequent antibody production. Danes e t al_ (1962) found t h a t the approximate i n h a l a t i o n l e t h a l dose o f a v i r u s aerosol of tick-borne e n c e p h a l i t i s v i r u s i n mice was e q u i v a l e n t to 10 to 40 i n t r a c r a n i a l LDg^ and that the i n c u b a t i o n p e r i o d was the same as that f o l l o w i n g i n t r a n a s a l i n s t i l l a t i o n of the v i r u s ; whereas the i n c u b a t i o n p e r i o d f o r the i n t r a p e r i t o n e a l and intravenous method o f i n o c u l a t i o n was l e s s . In the animals exposed to i n f e c t i o u s a e r o s o l s , v i r u s m u l t i p l i e d f i r s t i n the lungs where i t reached a t i t r e between 10^ and 10^ LD 5 Q/0.03 ml. On the t h i r d day i t appeared i n the blood and one day l a t e r i n the b r a i n where i t s con-c e n t r a t i o n i n c r e a s e d u n t i l the death o f the animal. High doses o f v i r u s com-pared to low doses tended to cause the v i r u s to appear e a r l i e r i n the various organs. Johnson (1964) i n his f l u o r e s c e n t - a n t i b o d y s t u d i e s of the patho-genesis of herpes v i r u s e n c e p h a l i t i s by various routes o f i n o c u l a t i o n found the f o l l o w i n g : a d r o p l e t c o n t a i n i n g 50,000 plaque forming un i t s o f v i r u s was necessary to cause 100 per cent m o r t a l i t y (as compared to 3 plaque forming units f o r the i n t r a c e r e b r a l and 14 plaque forming u n i t s f o r the i n t r a p e r i t o n e a l routes of i n o c u l a t i o n ) , mice remained well f o r four o r f i v e days then enceph-a l i t i s developed with median time o f death at f i v e days; organ t i t r a t i o n s were s i m i l a r to those obtained a f t e r i n t r a p e r i t o n e a l i n o c u l a t i o n but viremia was 1 3 2 9 lower ranging from l e s s than 10 to 10 " plaque forming units per ml. as 24 2 5 3 7 compared to 10 ' to 10 ' plaque forming units per ml. f o l l o w i n g the i n t r a -p e r i t o n e a l route o f i n o c u l a t i o n . A f t e r i n t r a c e r e b r a l i n o c u l a t i o n however, v i r u s was i r r e g u l a r l y recovered from the blood, l i v e r o r s p l e e n ; v i r u s t i t r e s rose r a p i d l y i n the b r a i n to 10^'^ plaque forming units per gm. by the t h i r d day r e s u l t i n g i n death o f the mice. I n i t i a l r a p i d m u l t i p l i c a t i o n i n the lungs was followed by hematogenous spread to the l i v e r and spleen on the second day. Again v i r u s was not detectable i n the c e n t r a l nervous system u n t i l the t h i r d day a f t e r i n o c u l a t i o n . Fluorescent f o c i were found i n lungs, but no antigen was found i n the mucosa o f the stomach or i n t e s t i n e s . F l u orescent c e l l s were not found w i t h i n the c e n t r a l nervous system before the fou r t h day. The patterns of fluorescence i n the c e n t r a l nervous system i n d i c a t e d that i n f e c t i o n of the nasal mucosa and submucosa t i s s u e s through the meninges i n t o the subarchnoid space r e s u l t e d i n widespread i n f e c t i o n o f the meninges. A l s o , i n f e c t i o n of o l f a c t o r y nerve f i b r e s with subsequent spread through the c r i b r i f o r m p l a t e to the o l f a c t o r y bulb was observed. N i r e_t al_ (1965) i n t h e i r study of the pathogenesis of West N i l e v i r u s a f t e r exposure o f mice to aerosols of the v i r u s s t a t e d that immediately a f t e r exposure, demonstrable q u a n t i t i e s o f v i r u s were d e t e c t a b l e i n the lungs only. I t was only a f t e r 48 hours that v i r u s appeared i n other organs. F l u o r -escent f o c i were not seen i n most s l i d e s of l i v e r , spleen and kidney t i s s u e taken from 0 to 144 hours a f t e r exposure to the a e r o s o l . On the other hand p o s i t i v e s t a i n i n g f o r antigen was noted i n macrophages i n a l l s e c t i o n s o f lung t i s s u e 24 hours a f t e r exposure. Antigen i n the b r a i n was observed at 48 hours and reached a maximum at 120 hours post-exposure. They p o s t u l a t e that i n v a s i o n of the c e n t r a l nervous system does not appear to stem from e a r l y m u l t i p l i c a t i o n i n the lung. No v i r u s was found i n c e r v i c a l lymph nodes or the blood before i t f i r s t appeared i n the b r a i n , which seems to r u l e out the hematogenous spread o f v i r u s from the lungs to the c e n t r a l nervous system. 2 5 Virus t i t r a t i o n s of various parts of the b r a i n seem to p o i n t to the i n v a s i o n o f the c e n t r a l nervous system along the o l f a c t o r y pathway. Conventional and f l u o r e s c e n t - a n t i b o d y s t a i n i n g of o l f a c t o r y bulbs tend to s u b s t a n t i a t e the above p o s t u l a t e . Henderson e_t al_ ( 1 9 6 7 ) studying the pathogenesis o f Semliki f o r e s t v i r u s i n hamsters i n d i c a t e d that v i r u s was f i r s t detected i n the o l f a c t o r y lobe at e i g h t hours post-exposure, at 1 4 hours i n the main p o r t i o n o f the b r a i n , at 2 6 hours i n the s p i n a l cord a f t e r aerosol exposure to 1 , 0 0 0 v i r a l u n i t s ( r e t a i n e d lung dose) of Semliki f o r e s t v i r u s . P a t h o l o g i c a l changes were a l s o observed i n the o l f a c t o r y mucosa and the l i v e r where an acute h e p a t i t i s was produced l a t e i n the course of the disease. No damage appeared i n lung e p i -thelium. They p o s t u l a t e the o l f a c t o r y pathway f o r the spread o f v i r u s to the c e n t r a l nervous system a f t e r aerosol i n o c u l a t i o n of Semliki f o r e s t v i r u s . Contact i n f e c t i o n between i n f e c t e d and non-infected hamsters was seen to occur. Because f l u o r e s c e n t - a n t i b o d y was not used i n t h i s study to reveal the presence of antigen, v i r a l m u l t i p l i c a t i o n i n various t i s s u e s could have been overlooked while using conventional h i s t o l o g i c a l techniques. M i l l e r ( 1 9 6 6 ) studying the h o s t - p a r a s i t e r e l a t i o n s h i p between Venezuelan equine e n c e p h a l i t i s v i r u s and white Carneau pigeons found that i n h a l a t i o n of v i r u s i n small p a r t i c l e s by normal birds r e s u l t e d i n i n f e c t i o n s when the inhaled dose i n one minute was more than 1 3 5 M I C L D ^ Q u n i t s (mouse i n t r a c r a n i a l l e t h a l doses 5 0 per cent end p o i n t ) . The i n f e c t i o n was charac-t e r i z e d by viremia o f two or three days duration followed by the development of serum n e u t r a l i z i n g a n t i b o d i e s . The presence o f the a n t i b o d i e s o f f e r e d p r o t e c t i o n a g a i n s t subsequent aerosol challenge. Virus was a l s o found to be present i n the o r a l c a v i t y , however a i r exhausted from the cage o f i n f e c t e d pigeons f a i l e d to cause i n f e c t i o n i n normal bi r d s upon i n t r o d u c t i o n o f the p o t e n t i a l l y i n f e c t i o u s a i r . N o n - s p e c i f i c r e s p i r a t o r y r e s i s t a n c e f a c t o r s were 26 invoked as the reason f o r f a i l u r e to i n f e c t normal b i r d s with the exhaust a i r o f i n f e c t e d b i r d s because i t was noted that subcutaneously i n o c u l a t e d bi r d s responded with an immediate occurrence o f viremia whereas a e r o s o l i z e d b i r d s showed only a p a r t i a l response with a delayed onset. I t was concluded that the c r i t i c a l f a c t o r i n the i n d u c t i o n o f viremia and s t i m u l a t i o n o f n e u t r a l i z i n g antibody formation was dose-dependent; the dose required neces-s a r i l y being g r e a t e r than the n o n - s p e c i f i c i n a c t i v a t i o n o f the v i r u s . Hruskova et al_ (1969), found that the i n h a l a t i o n L D 5 Q f o r guinea pigs to Venezuelan equine e n c e p h a l i t i s was eq u i v a l e n t to 3.5 guinea p i g i n t r a c e r e b r a l L D 5 Q and f o r mice; e q u i v a l e n t to 135 mouse i n t r a c e r e b r a l L D 5 Q or 100 guinea p i g L D ^ Q . L a t e r i n a flu o r e s c e n t - a n t i b o d y study Hruskova e t al_ (1969a), s t a t e d t h a t the course o f the i n f e c t i o n a f t e r aerosol challenge was acute and the nasal mucosa could be considered as: the s i t e o f primary v i r u s m u l t i p l i c a t i o n , the r e s e r v o i r f o r v i r e m i a , and the region from which the c e n t r a l nervous system was i n f e c t e d . Waldman e_t al_ (1970), f o l l o w i n g the antibody response i n humans when exposed to aerosols of i n f l u e n z a v i r u s vaccine showed that (1) the l e v e l o f n e u t r a l i z i n g antibody a f t e r aerosol immunization was about s i x times pre-immunization l e v e l s a year afterwards; (2) the response i n p a t i e n t s with c h r o n i c lung disease was as good or b e t t e r than normal i n d i v i d u a l s and they s u f f e r e d no ill e f f e c t s from aerosol procedures; (3) the antibody re-sponse i n c h i l d r e n three to ten years of age was o f a l e s s e r magnitude than those i n the seven to ten age group o r a d u l t s ; (4) the immunoglobulin c l a s s of n e u t r a l i z i n g a n t i b o d i e s was IgG i n the case o f serum and IgA i n the case o f s e c r e t i o n s ; (5) the p a r t i c l e s i z e o f the aerosol had a dramatic e f f e c t on the antibody response. The sm a l l e r p a r t i c l e s i z e s (1.5 micron) produced a be t t e r serum antibody (IgG) response; the l a r g e r p a r t i c l e s i z e s (40 - 100 27 microns) produced a b e t t e r s e c r e t i o n antibody (IgA) response; the e n t i r e spectrum of s i z e s (1.5 micron) produced a b e t t e r serum antibody (IgG) res-ponse; the l a r g e r p a r t i c l e s i z e s (40 - 100 microns) produced a b e t t e r s e c r e -t i o n antibody (IgA) response; the e n t i r e spectrum o f s i z e s (1.5 to 100 microns) stimu l a t e d sputum antibody responses. The s p e c i f i c aims were: (1) to deter-mine i f mice could be i n f e c t e d with Powassan v i r u s , a member of the Russian spring-summer complex of ti c k - b o r n e group B arboviruses by a means which d i d not introduce the v i r u s d i r e c t l y i n t o the bloodstream; (2) to provide information on the dose-response r e l a t i o n s h i p s using various routes o f i n o c u l a t i o n ; and (3) to determine the pathogenesis o f Powassan v i r u s i n mice a f t e r airborne i n f e c t i o n . MATERIALS AND METHODS  Vi rus Powassan v i r u s , prototype s t r a i n LB t h i r d mouse b r a i n passage, obtained from Dr. D. M. McLean, Department o f Micro b i o l o g y , U n i v e r s i t y o f B r i t i s h 7 8 Columbia, Vancouver 8, Canada e x h i b i t e d a t i t r e o f 10 ' mouse i n t r a c e r e b r a l l e t h a l doses 50 per cent end point (M I C L D ^ Q) per m i l l i l i t e r (ml.) when t i t r a t e d using three- to four-week-old Swiss Webster mice. Stocks o f v i r u s were prepared by i n o c u l a t i n g groups o f 50 s u c k l i n g mice i n t r a c e r e b r a l l y with 125 MICLD 5 Q i n 0.02 ml. of the above v i r u s , h a r v e s t i n g i n f e c t e d b r a i n t i s s u e , preparing 10 per cent homogenates and removing d i s r u p t e d c e l l u l a r debris by c e n t r i f u g a t i o n . Stocks of the LB prototype s t r a i n o f Powassan vir u s i n i t s fourth mouse bra i n passage were s t o r e d i n 0.1 ml. a l i q u o t s at -70°C and used f o r a l l experiments. In order to determine the optimum time at which to harvest stocks of Powassan v i r u s from i n f e c t e d s u c k l i n g mice, a growth curve was constructed i n the f o l l o w i n g manner: 65 s u c k l i n g mice were i n o c u l a t e d i n t r a c e r e b r a l l y with 125 MICLDr.n o f the Powassan v i r u s prototype s t r a i n LB t h i r d mouse b r a i n 28 passage. At 12 hour i n t e r v a l s f o r 144 hours, f i v e mice were s a c r i f i c e d , t h e i r b r a i n t i s s u e removed, pooled and placed i n a pre-weighed 10 to 12 ml. screw-capped s t e r i l e v i a l and s t o r e d at -70°C i n a Revco u l t r a low tempera-ture f r e e z e r (Model ULT 656 Revco Incorporated, D e e r f i e l d , Michigan). Sub-sequently the contents of each v i a l were placed i n a pre-cooled mortar at 4°C and ground with a p e s t l e . Two hundred r o t a r y movements were executed f o r each sample t r e a t e d . Microscopic examination o f samples o f the various homogenates i n d i c a t e d that the majority o f t i s s u e c e l l s were d i s r u p t e d a f t e r such treatment. A 10 per cent suspension i n E a r l e ' s balanced s a l t s o l u t i o n (EBSS) co n t a i n i n g 20 per cent heat i n a c t i v a t e d (56°C f o r one hour) c a l f serum, 200 i n t e r n a t i o n a l u n i t s per ml. p e n i c i l l i n and 200 micrograms per ml. streptomycin, pH 7.5, was prepared f o r each 12 hour time p e r i o d . C e l l u l a r debris was sedimented using the Beckman p r e p a r a t i v e u l t r a -c e n t r i f u g e Model L2HV using the type 30 f i x e d angle r o t o r (Beckman Instrument Incorporated, Palo A l t , C a l i f o r n i a ) at 10,000 x g. f o r 10 min. at 4 ° C The supernatant was decimally d i l u t e d i n EBSS. Each d i l u t i o n was examined f o r i n f e c t i v i t y by the i n t r a c e r e b r a l i n o c u l a t i o n o f three- to four-week-old weaned mice using 0.03 ml. per i n o c u l a t i o n and s i x mice per d i l u t i o n . The MICLDgg was c a l c u l a t e d by the method o f Reed and Muench (1938). Preparation o f Powassan v i r u s antigens, hemagglutination, and hema-g g l u t i n a t i o n - i n h i b i t i o n t e s t s were performed according to the procedures described i n Diagnostic Procedures f o r V i r a l and R i c k e t t s i a l I n f e c t i o n s , Hammon and Sather (1969). Fluorescent Antibody Hyperimmune serum to Powassan v i r u s was prepared i n the f o l l o w i n g manner: 1 0 7 * 7 MICLD 5 Q i n 5 ml. were i n o c u l a t e d i n t r a v e n o u s l y i n t o the mar-gi n a l ear vein of a 2.7 kilogram female r a b b i t . The primary i n o c u l a t i o n was followed by four s i m i l a r doses given i n t r a p e r i t o n e a l y spaced one week apart. 29 One week subsequent to the f i n a l i n o c u l a t i o n 50 ml. o f blood was removed from the marginal ear vein by p i e r c i n g the vein with a s t e r i l e l a n c e t and c o l l e c -t i n g extravasated blood i n a graduated c y l i n d e r . The blood was allowed to c l o t , rimmed with a s t e r i l e 1.0 ml. p l a s t i c p i p e t t e , placed i n a Mil n e r r e f r i -gerator Model RSS45 (Edward Milner Co., L t d . , Toronto, Canada) at 4°C over-n i g h t to allow f o r c l o t r e t r a c t i o n . Serum was removed with a Pasteur p i p e t t e , placed i n a c e l l u l o s e n i t r a t e c e n t r i f u g e tube and c e n t r i f u g e d i n the type 30 ro t o r using the Beckman L2HV pr e p a r a t i v e u l t r a c e n t r i f u g e a t 10,000 x g. f o r 10 minutes a t 4°C. To 20.0 ml. o f antiserum was added an equal q u a n t i t y o f 0.15 M sodium c h l o r i d e s o l u t i o n . A f l a s k c o n t a i n i n g the d i l u t e d serum was held at 4°C i n the Mi l n e r r e f r i g e r a t o r . Forty ml. o f satura t e d ammonium s u l f a t e was added to the d i l u t e d serum at a rate o f 60 drops per minute. During, and 30 minutes a f t e r the a d d i t i o n o f ammonium s u l f a t e to the d i l u t e serum the mix-ture was s t i r r e d using a Cenco (Central S c i e n t i f i c Co., Chicago) magnetic s t i r r i n g apparatus. The suspension was then poured i n t o pre-cooled c e l l u l o s e n i t r a t e c e n t r i f u g e tubes and held at 4°C f o r two hours. The p r e c i p i t a t e d gamma g l o b u l i n suspension was then c e n t r i f u g e d at 10,000 x g. f o r 10 minutes at 4°C. The supernatant was discarded and the p r e c i p i t a t e was washed with an a d d i t i o n a l 10 ml. o f c o l d h a l f - s a t u r a t e d ammonium s u l f a t e and r e c e n t r i fuged. The supernatant from the second c e n t r i f u g a t i o n was also discarded and the p r e c i p i t a t e was r e d i s s o l v e d i n 6.0 ml. o f s t e r i l e d e i o n i z e d water. The s o l u t i o n o f immune gamma g l o b u l i n was placed i n a length o f d i a l y s i s tubing o f 0.625 inch diameter ( i n f l a t e d ) ( F i s h e r S c i e n t i f i c Co.) and d i a l y z e d a g a i n s t phosphate b u f f e r e d s a l i n e pH 7.2 (P.B.S.) co n t a i n i n g 0.145 M sodium c h l o r i d e 0.01 M phosphate and c o n s i s t e d o f 8.5 g NaCl, 1.07 g Na 2HP0 4, and 0.39 g NaH 2P0 4. 2H 20'per l i t e r o f deio n i z e d wcjter. Four (100 volume) changes o f the external s o l u t i o n over a 64 hour p e r i o d was 30 s u f f i c i e n t to remove contaminating ammonium s u l f a t e when a 10.0 ml. sample o f the external s o l u t i o n f a i l e d to show the presence o f ammonium ion a f t e r being mixed with three drops o f Nessler's reagent. The immune g l o b u l i n was placed i n a s t e r i l e 10 to 12 ml. screw-capped v i a l and st o r e d at -20°C i n a Mil n e r f r e e z e r Model RSS 45 SF u n t i l conjugated with f l u o r e s c e i n i s o t h i o c y a n a t e (FITC) ( N u t r i t i o n a l Biochemicals Corp. C l e v e l a n d ) . P r i o r to FITC conjugation the ni t r o g e n content o f the g l o b u l i n was determined using the Perkin Elmer No. 240 elemental a n a l y z e r (Perkin-Elmer, Norwalk, C o n n e c t i c u t ) , Prezioso (1965). A commercial preparation o f r a b b i t gamma g l o b u l i n C0HN f r a c t i o n II 95 per cent p u r i t y ( N u t r i t i o n a l Biochemicals Corp., Cleveland) was used as a standard. The Powassan v i r u s hyperimmune r a b b i t gamma g l o b u l i n was conjugated with FITC according to the method out-l i n e d by Nairn (1969) and F o t h e r g i l l (1969). The removal o f unreacted f l u o r e s c e n t material was accomplished by gel f i l t r a t i o n with a Sephadex G-25 column i n phosphate b u f f e r e d s a l i n e . The dimensions o f the column were 15 m i l l i m e t e r s by 150 m i l l i m e t e r s . The Sephadex was washed three times with phosphate b u f f e r e d s a l i n e pH 7.2 to remove the ' f i n e s . ' A f t e r the t h i r d wash the gel beads were allowed to swell overnight i n the r e f r i g e r a t o r at 4°C. A s l u r r y o f the G-25 Sephadex i n phosphate b u f f e r e d s a l i n e was added to a K15/30 chromatographic column (Pharmacia Canada L t d . , Montreal, P.Q.) u n t i l the d e s i r e d bed height was reached. Excess b u f f e r was allowed to p e r c o l a t e through the column. The conjugate was added to the column when the b u f f e r became l e v e l with the bed s u r f a c e ; i t was allowed to ente r the bed completely before a d d i t i o n a l b u f f e r was added. C o l l -e c t i o n o f the e l u a t e began when the f i r s t v i s i b l e band o f dye began to leave the column and was disco n t i n u e d when the elu a t e became c o l o r l e s s . The e l u a t e was approximately twice the volume o f the conjugate which was added o r i g i n a l l y 3 1 to the Sephadex G-25 column. The volume o f the eluate was t h e r e f o r e reduced to the o r i g i n a l (6 ml.) with the a i d of u l t r a f i l t r a t i o n using a D i a f l o mem-brane XM 50, a Model 12 and Model 8MC u l t r a f i l t r a t i o n c e l l s (Amicon Corpora-t i o n , Lexington, Mass.) operated at 60 p s i . One hundred mi l l i g r a m s (mg.) /ml. o f l y o p h i l i z e d mouse b r a i n powder was added to the conjugate and s t i r r e d at 23°C f o r two hours. The mixture was subsequently c e n t r i f u g e d at 10,000 x g. f o r 10 minutes at 4°C. The p e l l e t was discarded and the supernatant con-jugate was t e s t e d f o r Powassan s p e c i f i c antibody using the n e u t r a l i z a t i o n t e s t (Nt) and h e m a g g l u t i n a t i o n - i n h i b i t i o n t e s t (HI) as p r e v i o u s l y noted (Hammon and Sather, 1969). L y o p h i l i z e d l i v e r and/or b r a i n powder was prepared i n the f o l l o w i n g manner: l i v e r t i s s u e was removed from ten healthy four-week-old mice. The t i s s u e was washed c l e a r o f blood with phosphate b u f f e r e d p h y s i o l o g i c a l s a l i n e pH 7.2 both before and a f t e r d i c i n g . An equal volume o f f r e s h c o l d physio-l o g i c a l s a l i n e was added to the d i c e d t i s s u e and the suspension was homogenized i n a high speed Waring blender f o r f i v e minutes at 4°C. The homogenate was t r e a t e d to three a l t e r n a t e c y c l e s o f washing with c o l d p h y s i o l o g i c a l s a l i n e and c e n t r i f u g a t i o n at 5000 x g. f o r 10 minutes at 4°C. The f i n a l c e n t r i f u -gation was at 10,000 x g. f o r 10 minutes at 4°C. The f i n e l y separated t i s s u e was resuspended f o r the l a s t time i n f r e s h volume o f s a l i n e , dispensed i n t o 2 ml. freeze-dry ampules and l y o p h i l i z e d using the V i r t i s Model 10-147-MR-BA F r e e z e - d r i e r ( V i r t i s Co., Gardiner, N.Y.) Routes o f I n f e c t i o n ( i ) I n t r a c e r e b r a l I n o c u l a t i o n Powassan v i r u s was assayed using the i n t r a c e r e b r a l l e t h a l dose 50 per cent end p o i n t i n three- to four-week-old mice. Decimal d i l u t i o n s o f v i r u s suspensions were i n o c u l a t e d i n t r a c e r e b r a l l y i n t o mice using 0.03 ml. o f inoculum per mouse and a t l e a s t f i v e mice per d i l u t i o n . Time to death was 3 2 recorded over an observation time o f ten days. The l e t h a l dose 5 0 per cent (LDgg) was determined s t a t i s t i c a l l y by the method o f Reed and Muench ( 1 9 3 8 ) . The strength o f the v i r u s suspensions t h e r e f o r e was recorded as mouse i n t r a -c erebral l e t h a l dose 5 0 per cent end p o i n t per m i l l i l i t e r (MIO - D^g/ml.). This was defined as: the minimum amount o f v i r u s suspension r e q u i r e d to cause death i n 5 0 per cent o f the mice i n o c u l a t e d by the i n t r a c e r e b r a l route using 0 . 0 3 ml. o f v i r u s suspension during an observation p e r i o d o f ten days. ( i i ) Intranasal I n s t i l l a t i o n Preparatory to studying the pathogenesis o f Powassan v i r u s i n mice i n f e c t e d using an i n f e c t i o u s a e r o s o l , i t was necessary to determine i f mice could be i n f e c t e d by a route which d i d not introduce v i r u s p a r t i c l e s d i r e c t l y i n t o the blood stream. Therefore, several mice were subjected to the i n t r a -nasal i n s t i l l a t i o n o f various concentrations o f Powassan v i r u s under l i g h t ether anaesthesia. A f t e r i t became apparent t h a t mice could indeed be i n f e c -ted by t h i s route the LDgQ was determined. A l s o , a l i m i t e d study o f v i r u s l e v e l s and time of appearance i n various t a r g e t organs was undertaken. The procedures used were as f o l l o w s : Powassan v i r u s L B s t r a i n i n 9 3 i t s fourth mouse b r a i n passage c o n t a i n i n g 1 0 MICLDgg per ml. was d i l u t e d - 1 - 8 decimally i n EBSS from 1 0 to 1 0 . Using nine mice per d i l u t i o n , one drop of v i r u s suspension (0.037nil •) was i n s t i l l e d i n t o the external nares o f each mouse with the a i d o f a c a l i b r a t e d Pasteur p i p e t t e . The course o f i n f e c t i o n , over a ten day p e r i o d was followed r e c o r -ding time to death. The mouse i n t r a n a s a l l e t h a l dose 5 0 per cent (M I N L D ^ Q ) was defined as the minimum amount o f Powassan v i r u s per ml. o f suspension which caused death i n 5 0 per cent of mice. The time o f appearance and l e v e l s o f Powassan v i r u s i n various t a r g e t t i s s u e s was s t u d i e d i n the f o l l o w i n g manner: 4 5 mice were i n o c u l a t e d with an i n t r a n a s a l i n s t i l l a t i o n o f 1 0 MINLDVn of Powassan v i r u s . Each 2 4 33 hours f o r seven days, f i v e mice were s a c r i f i c e d and the f o l l o w i n g t i s s u e s were pooled and examined f o r the presence of v i r u s : nasal e p i t h e l i u m ( T u r b i n a t e s ) , lung t i s s u e , blood, spleen and b r a i n t i s s u e . The lung, n a s a l , spleen and b r a i n t i s s u e s were washed fr e e o f blood both before and a f t e r d i c i n g . Ten per cent homogenates of the t i s s u e s were prepared i n EBSS. Deci-mal d i l u t i o n s were prepared o f the homogenate supernatants, and these were i n o c u l a t e d i n t r a c e r e b r a l l y i n 0.03 ml. amounts i n t o groups o f s i x mice per d i l u t i o n . Whole blood was d i v i d e d i n t o two a l i q u o t s . One a l i q u o t was allowed to c l o t and the serum was r e t a i n e d and s t o r e d at -20°C f o r l a t e r s e r o l o g i c a l examination f o r the presence o f Powassan v i r u s s p e c i f i c a n t i b o d i e s . The other a l i q u o t was l y s e d by freeze-thawing. Decimal d i l u t i o n s o f t h i s a l i q u o t were examined f o r i n f e c t i v i t y as above. ( i i i ) Subcutaneous In o c u l a t i o n ( i v ) Intravenous I n o c u l a t i o n ( v) I n t r a p e r i t o n e a l I n o c u l a t i o n ( v i ) Per 0s I n o c u l a t i o n The L D J J Q ' S by the above routes o f i n o c u l a t i o n were determined i n a s i m i l a r manner as f o l l o w s : mice under l i g h t anaesthesia were i n o c u l a t e d with decimal d i l u t i o n s of Powassan v i r u s LB s t r a i n i n i t s f o u r t h mouse b r a i n passage. 7 7fi This v i r u s stock contained 10 MICLDgg per ml. For the subcutaneous route o f i n o c u l a t i o n 0.03 ml. o f decimal d i l u -t i o n s o f Powassan v i r u s was i n o c u l a t e d subcutaneously, i n t o the dorsal region o f three- to four-week-old mice using nine mice per d i l u t i o n . For the intravenous route o f i n o c u l a t i o n 0.03 ml. o f decimal d i l u -t i o n s o f Powassan v i r u s was i n o c u l a t e d i n t o the l a t e r a l t a i l vein o f three-to four-week-old mice using nine mice per d i l u t i o n . For the i n t r a p e r i t o n e a l route of i n o c u l a t i o n 0.03 ml. o f decimal d i l u t i o n s o f Powassan v i r u s was i n o c u l a t e d through the ventral body wall and 34 i n t o the p e r i t o n e a l c a v i t y of three- to four-week-old mice using nine mice per d i l u t i o n . For the per os route of i n o c u l a t i o n 0.05 ml. o f decimal d i l u t i o n s of Powassan v i r u s was i n s t i l l e d d i r e c t l y i n t o the g a s t r o i n t e s t i n a l t r a c t using a s p e c i a l l y prepared 24 gauge needle 1.25 inches long which had been blunted by adding a drop o f brass to the end and g r i n d i n g the blunted needle smooth. The needle was introduced i n t o the mouth, down the esophagus and i n t o the stomach. Nine mice three- to four-weeks-old were used f o r each d i l u t i o n . The v i r u s d i l u e n t f o r t h i s experiment was s t e r i l e skimmed milk. A d u p l i c a t e s e r i e s of the above d i l u t i o n s were i n o c u l a t e d i n t r a c e r e b r a l l y i n t o three- to four-week-old mice using 0.03 ml., of inoculum and s i x mice per d i l u t i o n i n order to determine i f skimmed milk had any detrimental e f f e c t s on the v i a b i l i t y o f the v i r u s . The LD 5 0's by various routes were designated ajS f o l l o w s : mouse subcutaneous l e t h a l dose 50 per cent end p o i n t per ml. (MSCLDgQ/ml.), mouse intravenous l e t h a l dose 50 per cent end p o i n t per ml. (MIVLDgg/ml.), and mouse i n t r a p e r i t o n e a l l e t h a l dose 50 per cent end p o i n t per ml. (MIPLDgg/ml.). A l l LD^g's were determined using the method o f Reed and Muench (1938) and are r e l a t e d to the i n t r a c e r e b r a l L D 5 Q (MICLD 5 Q/ml.) i n three- to four-week-o l d mice. ( v i i ) Aerosol I n o c u l a t i o n The L D 5 0 by t h i s route of i n f e c t i o n was based on the t h e o r e t i c a l amount o f v i r u s i n h a l e d by exposed mice ( c a l c u l a t e d on the basis o f the minute r e s p i r a t o r y volume o f three- to four-week-old mice, Guyton (1947), the concentration o f v i r u s i n the a e r o s o l , and the length o f time of exposure of mice to the i n f e c t i o u s a e r o s o l ) . The actual r e t a i n e d dose o f v i r u s i n the lungs of i n d i v i d u a l mice a f t e r aerosol exposure o f groups of mice to a known conc e n t r a t i o n o f v i r u s f o r various periods o f time was a l s o examined. 35 During the f i r s t r e s p i r a t o r y i n o c u l a t i o n three- to four-week-old mice were exposed to aerosols o f Powassan v i r u s f o r two, f i v e and ten minutes using f i v e mice per exposure. A l l mice succumbed a f t e r seven or e i g h t days even when exposed f o r the minimal time of two minutes. During the second r e s p i r a t o r y exposure the f o l l o w i n g time p e r i o d s — 15, 30, 45, 60, 75, 90, 105 and 120 seconds -- using ten mice f o r each expo-sure p e r i o d were examined. E i g h t mice were held f o r ten days recording time to death. Two mice from each exposure p e r i o d were s a c r i f i c e d , lung t i s s u e s were removed, homogenized and pooled. C e l l u l a r d e bris was sedimented, d e c i -mal d i l u t i o n s o f the supernatants were prepared and assayed f o r i n f e c t i v i t y by i n t r a c e r e b r a l i n o c u l a t i o n o f t h r e e - to four-week-old mice. Subsequently, because recovery o f v i r u s from lung homogenates during the above exposures was too low to q u a n t i t a t e by the r e l a t i v e l y i n s e n s i t i v e LDgQ t i t r a t i o n method, mice were exposed to i n f e c t i o u s aerosols o f Powassan v i r u s f o r more extended periods o f time. Hence, mice were exposed f o r 5, 10 and 15 minutes using f i v e mice per exposure. Lung t i s s u e s were removed, weighed and 10 per cent homogenates were prepared. C e l l u l a r debris was sedimented by c e n t r i f u g a t i o n and the supernatants were q u a n t i t a t e d f o r v i r u s content. Aerosol Techniques A s p e c i a l f a c i l i t y (Figure 1) was designed i n order to expose experimental animals to dynamic aerosols of Powassan v i r u s plus study the aerosol c h a r a c t e r i s t i c s o f microorganisms while p r o v i d i n g a maximum o f pro-t e c t i o n to personnel engaged i n various experimental aerosol techniques. The f a c i l i t y c o n s i s t e d o f a s u i t e o f l a b o r a t o r i e s provided with a conditioned a i r supply which remained negative i n pressure with respect to the r e s t o f the l a b o r a t o r y b u i l d i n g ( i . e . , a l l a i r flow was c o n t r o l l e d so that when a breach o f the aerosol s u i t e was made by opening a door o r autoclave, the a i r flow was d i r e c t e d i n t o the aerosol s u i t e r a t h e r than the r e v e r s e ) . Furthermore 36 a l l a i r l e a v i n g the s u i t e passed through banks of u l t r a v i o l e t lamps which emitted 1600 micro watts per square centimeter (1600 microwatts/cm ) at the germicidal wave length o f 2537 Angstrom u n i t s . Subsequently a i r was then d i r e c t e d through an a i r i n c i n e r a t o r which was operated at a temperature at the bottom of a 66 f o o t high stack o f 650°F one f o o t above the flame, to 450°F at the top. The length of time r e q u i r e d f o r a p a r t i c l e to t r a v e r s e the stack was approximately 80 seconds. The e f f i c a c y of the i n c i n e r a t o r to take care o f r e s i d u a l i n f e c t i o u s aerosols was t e s t e d using B a c i l l u s s u b t i l i s  var n i g e r spores (Davids, 1971 unpublished data). Within the f a c i l i t y was l o c a t e d an i n s u l a t e d temperature and r e l a t i v e humidity c o n t r o l l e d chamber. I t was t h i s chamber that housed the aerosol decay t e s t apparatus (Figure 1, P l a t e 1) and a l s o the aerosol animal exposure u n i t ( P l a t e 2). A l l l a b o r a t o r i e s w i t h i n the aerosol s u i t e were pro-vided with r e g u l a r , emergency and u l t r a v i o l e t germicidal lamps mounted i n the c e i l i n g . Each l a b o r a t o r y had c o n t r o l s f o r u l t r a v i o l e t lamps but the regular and emergency ( i n case o f power f a i l u r e ) l i g h t s were c o n t r o l l e d by one main switchbox j u s t o u t s i d e the entry door to the aerosol s u i t e . Communication to a l l the microbiology l a b o r a t o r i e s o u t s i d e the s u i t e was provided by a vocal and warning l i g h t system so that i n case of accident proper recovery procedures could be i n s t i t u t e d immediately. Two autoclaves were provided, one small u n i t , 18" x 18" x 36" which was s i t u a t e d between the aerosol s u i t e and the hallway of the general microbiology l a b o r a t o r i e s . A l l m a t e r i a l s r e q u i r i n g wash-up were autoclaved and presented through t h i s autoclave. The other a u t o c l a v e , a l a r g e model, 24" x 36" x 60" was used to autoclave a l l disposable material ( p l a s t i c a n i -mal cages, dead i n f e c t i o u s l a b o r a t o r y animals, e t c . ) . A l l doors s e p a r a t i n g the s u i t e from the general l a b o r a t o r i e s were equipped with s p e c i a l l y designed s e a l s . Access and egress were accomplished through door 'A'; experimental animals and other s u p p l i e s were admitted 37 through door 'B1 and s t e r i l e disposable material was e x i t e d through t h i s door as w e l l . Each l a b o r a t o r y w i t h i n the s u i t e was s u p p l i e d with f i l t e r s t e r i -l i z e d and humidity c o n d i t i o n e d breathing a i r which was d e l i v e r e d to over-head bayonet-type hose connectors. I n v e s t i g a t o r s , t h e r e f o r e , using s p e c i a l l y designed p o s i t i v e pressure p l a s t i c hoods (Pla t e 3) were able to attach them-selves by long f l e x i b l e hoses to a separate s t e r i l e breathing a i r supply. Decontamination showers were provided and used before personnel returned to the general l a b o r a t o r y area. Showers were a l s o equipped with u l t r a v i o l e t germicidal lamps f o r shower s t a l l decontamination. A d r e s s i n g procedure was observed when the aerosol s u i t e was to be used. S t r e e t c l o t h e s were removed i n the outside d r e s s i n g room, l a b o r a -tory clothes ( c o v e r a l l s , gown, footwear, etc.) were put on i n the i n s i d e d r e s s i n g room. The reverse procedure i n t e r r u p t e d by showering was observed when l e a v i n g the s u i t e . Modified H o r s f a l l - t y p e units ( P l a t e 4) were i n s t a l l e d i n the a n i -mal h o l d i n g room. Animals which had been exposed to a e r o s o l s were placed i n p l a s t i c disposable cages w i t h i n the H o r s f a l l - t y p e u n i t s . A i r e n t e r i n g and l e a v i n g the H o r s f a l l - t y p e u n i t s was f i l t e r s t e r i l i z e d , passed through banks of u l t r a v i o l e t germicidal lamps and the a i r i n c i n e r a t o r before release to o u t s i d e . A l l d i s s e c t i o n s and other manipulations with regard to i n f e c t e d animals were confined to the aerosol s u i t e . ( i ) Powassan Virus Decay Studies Various decontamination procedures were examined f o r purposes o f s a f e t y . The e f f e c t i v e n e s s o f the u l t r a v i o l e t germicidal lamps f o r the decon-tamination o f a c c i d e n t a l s p i l l s was examined i n the f o l l o w i n g manner: 8 0 Powassan v i r u s c o n t a i n i n g 1 0 MICLD,-n/ml. i n EBSS was maintained both i n 38 p the dark and under u l t r a v i o l e t i r r a d i a t i o n at 20 microwatts/cm which was the minimum amount of r a d i a t i o n encountered on the f l o o r i n the aerosol s u i t e when a l l the u l t r a v i o l e t lamps were a c t i v a t e d . Thin f i l m s (2 mm. deep) o f Powassan v i r u s suspensions were placed i n 100 mm. p e t r i dishes. Samples o f the suspensions were taken at 30 minute i n t e r v a l s , under the dark c o n d i t i o n and at 15 minute i n t e r v a l s under the u l t r a v i o l e t r a d i a t i o n l i g h t c o n d i t i o n f o r a t o t a l elapsed time o f 180 minutes and assayed f o r v i r u s i n f e c t i v i t y using three- to four-week-old mice. A decay curve o f Powassan v i r u s under the two c o n d i t i o n s was p l o t t e d . The b i o l o g i c a l decay o f Powassan v i r u s at low, intermediate and high r e l a t i v e humidity (RH) and a constant temperature o f 21°C (69.8°F) was examined using the 500 l i t r e r o t a t i n g aerosol drum (Goldberg e t al_, 1958). o o Powassan v i r u s suspensions c o n t a i n i n g 10 MICLDgg per ml. under c o n t r o l l e d RH and temperature c o n d i t i o n s were disseminated f o r ten minutes i n t o the 500 l i t r e r o t a t i n g ( 3 - 4 rpm) s t a i n l e s s s t e e l drum using a C o l l i son atomizer operated at 26 pounds per square i n c h . This atomizer generates aerosol p a r t i c l e s i n the 1 to 5 micron range at the above oper a t i n g pressures (Green and Lane, 1964). Samples were taken every hour f o r a t o t a l elapsed time o f f i v e hours using 12/30 a l l glass impingers (AGI 12/30) (Brachman e t al_, 1964) con-t a i n i n g 10 ml. o f EBSS complete with ten per cent heat i n a c t i v a t e d c a l f serum. The impingers were operated at 12.5 l i t r e s per minute and the sample time was one minute i n d u r a t i o n . A l l impinger samples were t r a n s f e r r e d to 10 ml. s t e r i l e screw-capped ampules and s t o r e d a t -70°C u n t i l the completion of the i n v e s t i g a t i o n . Dissemination f l u i d s (both before and a f t e r atomizer r e f l u x i n g ) and impinger f l u i d s were examined f o r i n f e c t i v i t y using mice. Three experiments were completed f o r each RH range examined ( i . e . , 39 20 per cent RH, 50 per cent RH, 80 per cent RH at 21°C). The b i o l o g i c a l decay of Powassan v i r u s aerosols i n the presence o f germicidal u l t r a v i o l e t r a d i a t i o n was determined using the f o l l o w i n g tech-o niques and equipment: Powassan vi r u s c o n t a i n i n g 10 MIGLDgg/ml. was d i s -persed i n t o an aerosol drum using a C o l l i s o n atomizer operated at 26 p s i . The v i r u s aerosols were aged w i t h i n the drum f o r three hours. Samples of the aerosol were taken at p r e s c r i b e d i n t e r v a l s using an AGI 12/30 operated at 12.5 l i t r e s per minute f o r one minute. Impingers contained 10 mis. o f EBSS with 10 per cent heat i n a c t i v a t e d c a l f serum added. Three experiments under an u l t r a v i o l e t r a d i a t i o n i n t e n s i t y of 400 microwatts/cm average plus three under the dark c o n d i t i o n were completed. A l i q u o t s of the various dissemination and impinger f l u i d s from r e l a t e d time periods f o r the two experimental c o n d i t i o n s were assayed f o r v i r u s i n f e c t i v i t y i n the usual manner. ( i i ) C r o s s - I n f e c t i o n T h i r t y mice, three- to four-weeks o l d , were exposed to Powassan v i r u s aerosols and held f o r one day i n H o r s f a l l - t y p e units to allow grooming p r a c t i c e s . An equal number o f marked uninfected mice were placed with the exposed group and the colony was maintained f o r 30 days a l l o w i n g intimate contact to take place between i n f e c t e d and non-infected mice. ( i i i ) Aerosol Exposure of Mice A r e s t r a i n i n g cage hol d i n g f i v e mice was designed which o r i e n t e d the mice i n the d i r e c t i o n o f an incoming i n f e c t i o u s aerosol ( P l a t e 2). The i n f e c t i o u s aerosol was generated using a C o l l i s o n atomizer operated at 26 psi and was d i r e c t e d i n t o a mixing tube (equipped with a water manometer so that the atmospheric over or under pressure could be c o n t r o l l e d using a separate c o n d i t i o n e d a i r supply) j u s t p r i o r to being admitted to the mouse exposure chamber. Residual aerosol (aerosol d i s t a l to the exposure chamber) was removed under vacuum, f i l t e r e d , subjected to u l t r a v i o l e t i r r a d i a t i o n and a i r i n c i n e r a t i o n before being r e l e a s e d to the o u t s i d e . I n v e s t i g a t o r s wore p r o t e c t i v e c l o t h i n g plus the p o s i t i v e pressure hood. Mice were exposed to an i n f e c t i o u s aerosol of Powassan v i r u s i n the f o l l o w i n g manner: under l i g h t ether anaesthesia f i v e mice at a time 5 4 were exposed to an aerosol c o n t a i n i n g 10 M I C L D 5 0 / l i t r e f o r f i v e minutes u n t i l 50 mice had been exposed. The f o l l o w i n g t i s s u e s were removed from four of the f i v e mice s a c r i f i c e d f o r each 24 hour time p e r i o d post exposure f o r a t o t a l elapsed time of e i g h t days f o r the purpose o f t i s s u e v i r u s con-tent determinations: 1) nasal e p i t h e l i u m (Turbinates) 2) lung t i s s u e 3) thymus t i s s u e 4) whole blood 5) sera 6) spleen t i s s u e 7) l i v e r t i s s u e 8) kidney t i s s u e 9) g a s t r o i n t e s t i n a l t r a c t t i s s u e 10) s t r i a t e d muscle t i s s u e 11) c a r d i a c muscle t i s s u e 12) smooth muscle t i s s u e 13) b r a i n t i s s u e T i s s u e s , with the exception o f whole blood and serum, were removed, pooled, washed f r e e o f blood with s t e r i l e b u f f e r e d p h y s i o l o g i c a l s a l i n e pH 7.0, weighed, d i c e d , washed three more times with s a l i n e and homogenized using s t e r i l e , Byrex brand glass t i s s u e grinders ( F i s h e r S c i e n t i f i c Co.) at 2°C. Ten per cent suspensions were c l e a r e d o f c e l l u l a r debris by c e n t r i f u g a t i o n as noted e a r l i e r . Decimal d i l u t i o n s o f the t i s s u e homogenate supernatants 41 were examined f o r v i r u s by the i n t r a c e r e b r a l i n o c u l a t i o n o f mice. Blocks o f the above t i s s u e s were taken from the remaining mouse and saved f o r micro-s c o p i c examination. Histopathology Mouse t i s s u e s were subjected to a v a r i e t y o f h i s t o l o g i c a l techniques i n an endeavour to p i c t u r e o v e r t pathology caused by Powassan v i r u s and als o to confirm the types o f t i s s u e c e l l s i n which the v i r u s was r e p l i c a t i n g . ( i ) H i s t o l o g i c a l Techniques Duplicate blocks o f the various types o f i n f e c t e d t i s s u e s were pro-cessed i n order t h a t they could be examined using f l u o r e s c e n t antibody (Powassan v i r u s s p e c i f i c FITC conjugated r a b b i t gamma g l o b u l i n ) and/or e l e c -tron microscope techniques. ( i i ) S e c t i o n i n g Techniques Blocks of t i s s u e s were removed from i n f e c t e d mice and snap-frozen i n l i q u i d n i t r o g e n . Tissue blocks were then placed i n v i a l s and s t o r e d a t -70°C u n t i l s e c t i o n e d . Tissues were a p p l i e d to the chuck of a SLEE (South London E l e c t r i c a l Equipment Co. Ltd.) Model HRM microtome c r y o s t a t using a drop of s a l i n e . With the temperature o f the c r y o s t a t s e t at -22°C, ribbons o f s e c t i o n s were cut at s i x microns thickness employing the a n t i r o l l bar. Sections were picked up and placed on c l e a n , cooled (-22°C) 22 x 40 mm. c o v e r s l i p s using a camel h a i r brush. Tissue was f i x e d to the c o v e r s l i p by applying a f i n g e r to the underside of the c o l d c o v e r s l i p d i r e c t l y beneath the t i s s u e s e c t i o n . Sections were a i r d r i e d a t 37°C f o r one hour then s t a i n e d with f l u o r e s c e n t - a n t i b o d y . Tissue s e c t i o n s scheduled f o r hema-t o x y l i n and ebsin (H and E) s t a i n i n g were placed d i r e c t l y i n t o the s t a i n f o r two minutes. A m o d i f i c a t i o n o f the H and E s t a i n as described by Humason (1967) was used to s t a i n snap-frozen s e c t i o n s o f mouse t i s s u e . Sections 42 of various t i s s u e were t r e a t e d i n the f o l l o w i n g manner: ( 1) H a r r i s ' hematoxylin - two minutes; ( 2) wash i n tap water; ( 3) dip i n a c i d - a l c o h o l ; ( 4) wash i n tap water; ( 5) dip i n Scott's s o l u t i o n u n t i l blue; ( 6) check m i c r o s c o p i c a l l y f o r proper d i f f e r e n t i a t i o n ; ( 7 ) a l c o h o l - e o s i n three to f i v e minutes; ( 8) wash i n tap water; ( 9) ten dips i n 95 per cent ethyl a l c o h o l ; (10) ten dips i n 99 per cent eth y l alcohol (molecular s i e v e s ) ; (11) ten dips i n 99 per cent ethyl alcohol (molecular s i e v e s ) ; (12) xylene three minutes; (13) mount with permount. ( i i i ) Fluorescence Microscope Techniques For f l u o r e s c e n c e microscopy, s e c t i o n s were t r e a t e d according to the f o l l o w i n g p r o t o c o l : a) Powassan v i r u s s p e c i f i c FITC conjugated r a b b i t gamma g l o b u l i n , showing s p e c i f i c s t a i n i n g ( d i r e c t s t a i n i n g method). b) Unconjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , washed, con-jugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n showing no s t a i n i n g ( b l o c k i n g t e s t ) . c) Unconjugated n o n - s p e c i f i c gamma g l o b u l i n ( c o n t r o l g l o b u l i n ) , washed, con-jugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , showing s p e c i f i c s t a i n i n g ( c o n t r o l b l o c k i n g t e s t ) . d) Unconjugated Powassan v i r u s s p e c i f i c r a b b i t antiserum, washed, conjugated goat a n t i - r a b b i t g l o b u l i n , showing s p e c i f i c s t a i n i n g ( i n d i r e c t s t a i n i n g method). 43 e) Unconjugated n o n - s p e c i f i c r a b b i t antiserum, washed, conjugated goat a n t i -r a b b i t g l o b u l i n showing no s t a i n i n g (antiserum c o n t r o l ) . Tissues processed f o r fl u o r e s c e n c e examination were viewed using a L e i t z Wetzlar Ortholux fluorescence microscope equipped with an Osram HBO 200 high pressure mercury lamp a f t e r which a heat f i l t e r , UG1 u l t r a v i o l e t transmission f i l t e r , a BG 38 red suppression f i l t e r plus two K430 u l t r a v i o l e t suppression f i l t e r s attached to an e i g h t power p e r i p l a n b i n o c u l a r . A D.O. 80 L e i t z dark f i e l d condenser was used f o r fl u o r e s c e n c e microscopy. Sections were photographed with a Zeiss Contarex S 35 mm. camera equipped with interchangeable backs f o r black/white and colour photography. Kodak Plus X, ASA 125, was used f o r black/white photographs. Kodak high speed Ektachrome, ASA 160 was used f o r colour photographs. Exposure times were determined using a Zeiss Ikophot M exposure meter equipped with a micro-scope adapted s l i d e - i n sensor which i n d i c a t e d l i g h t i n t e n s i t i e s d i r e c t l y from the s e c t i o n . H and E s t a i n e d s e c t i o n s were observed using the same microscope equipped with a f i v e v o l t tungsten l i g h t source and a L e i t z No. 601 swing-out b r i g h t f i e l d condenser. ( i v ) E l e c t r o n Microscope Techniques Various types o f t i s s u e from various elapsed time periods were placed i n a glass p e t r i dish under 2 per cent glutaraldehyde i n phosphate b u f f e r pH 7.4. The t i s s u e was s l i c e d i n t o cubes 1 mm. on the edge. Tissues were f i x e d f o r two hours i n 2 per cent glutaraldehyde then two hours i n a 1 per cent osmium t e t r o x i d e p r e p a r a t i o n . The f o l l o w i n g protocol was followed f o r the s t a i n i n g and embedding o f the various t i s s u e s : ( 1) t i s s u e washed f i v e times with 0.1 M phosphate and 0.2 M suc-rose b u f f e r pH 7.4; ( 2) f i x e d with 1 per cent osmium t e t r o x i d e i n 0.1 M phosphate 0.2 M sucrose b u f f e r f o r two hours; ( 3) wash f i v e times 0.1 M phosphate, 0.2 M sucrose b u f f e r ; ( 4) dehydrate i n 30 per cent ethanol 10 minutes; ( 5) dehydrate i n 70 per cent ethanol 10 minutes; ( 6) dehydrate i n 80 per cent ethanol 10 minutes; ( 7) dehydrate i n 95 per cent ethanol 10 minutes; ( 8) dehydrate i n absolute ethanol (with molecular s i e v e s ) 10 minutes; ( 9) dehydrate i n Propylene oxide (with molecular s i e v e s ) 15 minutes; (10) dehydrate i n Propylene oxide (with molecular s i e v e s ) 15 minutes; (11) dehydrate i n Propylene oxide and Epon 812 r a t i o 4:1 one hour; (12) dehydrate i n Propylene oxide and Epon 812 r a t i o 1:1 one hour; (13) embed i n Epon 812. The formulation of the epoxy r e s i n was as f o l l o w s : Mixture A: Epon 812 ( F i s h e r S c i e n t i f i c ) 62 ml. Dodecenyl s u c c i n i c anhydride 100 ml. Mixture B: Epon 812 100 ml. Nadic methyl anhydride 89 ml„ The above mixtures were compounded i n a r a t i o o f 4A : 6B, 1.5 per cent DMP (2-, 4-, 6 - t r i dimethyl aminomethy1 phenol) a c c e l e r a t o r was added to the epoxy r e s i n . Cubes o f t i s s u e were d r i e d thoroughly and placed i n the extreme end o f beam capsules ( F i s h e r S c i e n t i f i c Co.) epoxy r e s i n was added using a 10 ml. syringe equipped with a 14 gauge needle. Capsules were placed i n a 60°C oven f o r 48 hours. Sections were cut using an LKB Ultrotome type 4801A (Stockholm, Sweden). Ribbons of s e c t i o n s were a p p l i e d to copper e l e c t r o n microscope g r i d s and p o s t - s t a i n e d with uranyl acetate (5 gms. o f uranyl acetate in 100 ml. 45 d i s t i l l e d water, mix 1 ml. of the l a t t e r with 1 ml. of absolute a l c o h o l ) and lead c i t r a t e (0.04 gms. NaOH i n 50 ml. d i s t i l l e d water, mix 25 ml. o f the l a t t e r with 0.125 gms. of lead c i t r a t e ) . Grids were viewed using an AEI 801 high r e s o l u t i o n e l e c t r o n microscope (AEI S c i e n t i f i c Apparatus L t d . , Harlow, Essex, England). Photomicrographs were taken using I I f o r d 3k" x 3V p l a t e s and LR I l f o p r i n t LR 4 IP photographic paper. RESULTS  Vi rus Figure 3 represents the incre a s e i n Powassan v i r u s content i n the br a i n t i s s u e of three-day-old s u c k l i n g mice, at 12 hour i n t e r v a l s a f t e r the i n t r a c e r e b r a l i n o c u l a t i o n o f 125 MICLD 5 0 of Powassan v i r u s . Optimum concen-t r a t i o n s of v i r u s i n the mouse b r a i n occurred at between f i v e and s i x days, g where the t i t r e was always approximately 10 MICLDgg. I f t i s s u e was t i t r a -ted a f t e r the death of the animal, recovery of v i r u s was u s u a l l y l e s s than the optimum. Sucrose-acetone e x t r a c t s o f Powassan v i r u s - i n f e c t e d s u c k l i n g mouse b r a i n , r o u t i n e l y provided Powassan v i r u s hemagglutinin with an average r e c i -procal hemagglutinin t i t r e of from 256 to 512 at optimum pH, u s u a l l y 6.4 Fluorescent Antibody Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n a f t e r conjugation with FITC, p u r i f i c a t i o n and concentration using G25 Sephadex and D i a f l o u l t r a -4 f i l t r a t i o n r e s p e c t i v e l y n e u t r a l i z e d 10 M I C L D , - Q/0.03 ml. and a r e c i p r o c a l hemagglutination i n h i b i t i o n t i t r e o f 1024 f o r e i g h t hemagglutinating doses of v i r u s . Routes o f I n f e c t i o n Table 4 reveals the minimum dose o f Powassan v i r u s per ml. re q u i r e d 46 to cause death i n 50 per cent of i n f e c t e d animals a f t e r i n f e c t i o n by various routes. I t should be noted that the same dose of Powassan v i r u s i s required to cause i n f e c t i o n and death i n mice regardless of whether the v i r u s i s administered by the i n t r a c e r e b r a l or the intravenous route o f i n o c u l a t i o n . Mice could not be i n f e c t e d by the i n t r o d u c t i o n o f high concentra-tions ( 1 0 6 , 5 M I C L D ^ Q ) of Powassan v i r u s d i r e c t l y i n t o the g a s t r o i n t e s t i n a l t r a c t (see P l a t e 5 ) , using a s p e c i a l l y prepared needle to l i m i t the advent o f a c c i d e n t a l t i s s u e i n o c u l a t i o n . Intranasal I n s t i l l a t i o n A f t e r i t was determined that mice could be i n f e c t e d with Powassan vi r u s by the i n t r a n a s a l i n s t i l l a t i o n of suspensions of v i r u s , various key mouse t i s s u e s were examined f o r l e v e l s of Powassan v i r u s at various time i n t e r v a l s a f t e r i n o c u l a t i o n . This p o r t i o n of the study was s t a r t e d and completed at the Univer-s i t y . Intranasal i n s t i l l a t i o n of the v i r u s was used i n s t e a d of aerosol inocu-l a t i o n because the U n i v e r s i t y of B r i t i s h Columbia d i d not have an aerosol safe f a c i l i t y . Figure 4 reveals the l e v e l s o f Powassan v i r u s i n the t u r b i n a t e s (nasal e p i t h e l i u m ) , lung t i s s u e , blood, spleen t i s s u e , and b r a i n t i s s u e a f t e r the i n t r a n a s a l i n s t i l l a t i o n o f 10 MINLDgg of Powassan v i r u s . I t should be noted t h a t 3 log of v i r u s was recovered from the lung a f t e r i n o c u l a t i o n whereas undetectable l e v e l s were observed i n other t i s s u e s immediately a f t e r i n o c u l a t i o n . Hencej a s i z e a b l e inoculum was introduced d i r e c t l y i n t o the mouse lung. As mentioned above, high concentrations o f Powassan v i r u s introduced d i r e c t l y i n t o the g a s t r o i n t e s t i n a l t r a c t o f mice could not i n i t i a t e an i n f e c t i o n . This would then i n d i c a t e that the mice sub-j e c t e d to i n t r a n a s a l i n s t i l l a t i o n of Powassan v i r u s suspensions became i n f e c -ted by way o f the lung. Virus l e v e l s i n the nasal e p i t h e l i u m began to r i s e 4 7 immediately; 2 4 hours a f t e r i n o c u l a t i o n 2 . 6 log o f Powassan v i r u s appeared i n t h i s t i s s u e . There was a l a g of between one to two days before a p p r e c i a b l e l e v e l s o f v i r u s were detectable i n the blood. On the second day 3 . 2 l o g o f Powassan v i r u s appeared i n the blood. Between days two and three a f t e r inocu-l a t i o n , l e v e l s o f Powassan v i r u s i n the spleen rose a p p r e c i a b l y . As soon as appreciable q u a n t i t i e s o f v i r u s were detected i n the blood, comparable l e v e l s were detected i n the b r a i n . M u l t i p l i c a t i o n w i t h i n the b r a i n t i s s u e was observed from the second day a f t e r i n o c u l a t i o n u n t i l a maximum t i t r e of 9 . 2 l o g was a t t a i n e d a t seven days. Aerosol I n o c u l a t i o n The minimum dose o f Powassan v i r u s (administered by the aerosol route of i n o c u l a t i o n ) r e q u i r e d to i n i t i a t e i n f e c t i o n and death i n 5 0 per cent of mice exposed was found to be 6 4 0 M I C L D , - Q. Figure 5 reveals the apparent r e t a i n e d dose received by mice when they were exposed to Powassan v i r u s aerosols f o r periods up to 1 5 minutes. Recovery of v i r u s from lung homogenates was not p o s s i b l e f o r ex-posure times o f l e s s than four to f i v e minutes. Furthermore r e t a i n e d dosage o f Powassan v i r u s appeared to be f a r lower than the c a l c u l a t e d t h e o r e t i c a l minimum aerosol l e t h a l dose 5 0 per cent. A 2 0 minute exposure was attempted, but atomizer f l u i d became ex-hausted before the 2 0 minute exposure time was completed. It i s i n t e r e s t i n g to note that the r e t a i n e d dose approximately doubles f o r each f i v e minute extension o f the exposure time. Powassan Virus Decay Studies In the i n t e r e s t o f personal s a f e t y and al s o i n an endeavour to l e a r n a l i t t l e about the aerosol c h a r a c t e r i s t i c s o f Powassan v i r u s a rep-r e s e n t a t i v e o f the group B arboviruses as compared with the aerosol charac-48 t e r i s t i e s o f Semliki Forest v i r u s , a r e p r e s e n t a t i v e o f the group A arbo-viruses which was s t u d i e d e a r l i e r (Gaunt, 1968, 1969), various aspects o f the b i o l o g i c a l decay of Powassan v i r u s under a v a r i e t y of c o n d i t i o n s were examined. The r e s u l t s o f these i n v e s t i g a t i o n s are presented below. Figure 6 presents data on the e f f e c t exerted on shallow (2 mm.) suspensions o f Powassan v i r u s when they are subjected to u l t r a v i o l e t i r r a -d i a t i o n . 'Normal' decay of Powassan v i r u s suspensions maintained i n the dark at 21°C i n a balanced s a l t s o l u t i o n c o n t a i n i n g serum was approximately 1 l o g per ml. o f suspension a f t e r three hours. Whereas Powassan v i r u s sus-pensions subjected to u l t r a v i o l e t i r r a d i a t i o n a t an i n t e n s i t y of 20 micro-2 watts/cm. decayed r a p i d l y so that a f t e r 90 minutes o f exposure v i r u s could not be detected i n the suspensions. The curves are derived from data obtained from three separate experiments f o r each c o n d i t i o n . These data are presented n u m e r i c a l l y i n Table 8. The b i o l o g i c a l decay o f Powassan v i r u s aerosols under various con-d i t i o n s o f r e l a t i v e humidity and a constant temperature i s i l l u s t r a t e d i n Figure 7. The numerical p r e s e n t a t i o n o f these data appear i n Table 9. The p h y s i c a l decay o f the aerosol as revealed by the B a c i l l u s sub- t i l us spore was e s s e n t i a l l y zero. I t can be s t a t e d that Powassan v i r u s aerosols are s l i g h t l y more s t a b l e a t low r e l a t i v e humidity than at high or intermediate r e l a t i v e h u m i d i t i e s . Very l i t t l e l o s s of v i a b i l i t y occurs a f t e r one hour o f cloud age. By f a r the g r e a t e s t l o s s of v i a b i l i t y occurs w i t h i n the f i r s t hour, i n f a c t , w i t h i n the f i r s t few minutes a f t e r aerosol dissemi-nation . Samples o f spray f l u i d were examined both before and a f t e r dissemi-nation i n order to determine i f the r e f l u x i n g a c t i o n o f the C o l l i s o n atomizer 49 had a detrimental e f f e c t on the v i a b i l i t y of the v i r u s . On the average, spray f l u i d s a f t e r dissemination showed s l i g h t l y higher l e v e l s of v i r u s con-tent than spray f l u i d s assayed before dissemination of the a e r o s o l . Points on the curves are i n terms of mean M I C L D 5 0 per ml. o f impin-ger or c o l l e c t i n g f l u i d * These values can be converted to M I C L D ^ Q per l i t r e o f a i r by m u l t i p l y i n g by 0.8. Figure 8 presents the decay data i n terms of r e l a t i v e humidities hour by hour; Figure 9 and Table 10 present data on the b i o l o g i c a l decay of Powassan v i r u s aerosols i n the dark and under u l t r a v i o l e t i r r a d i a t i o n . C r o s s - I n f e c t i o n Table 5 reveals the r e s u l t s observed when 30 non-infected mice were placed with 30 mice which had been exposed to an i n f e c t i o u s Powassan vi r u s a e r o s o l . A l l o f the mice that were subjected d i r e c t l y to the i n f e c t i o u s aerosol were dead by the 11th day post-exposure. Of the 30 non-infected mice 13 succumbed to Powassan vi r u s i n f e c t i o n contracted because of close contact with the i n f e c t e d group. The n o n - s p e c i f i c deaths were due to two mice which s u f f o c a t e d because they became caught i n the cage a i r exhaust p o r t . These non-s p e c i f i c deaths occurred too e a r l y to determine i f they had r e c e i v e d a s u f f i -c i e n t dose o f v i r u s to become i n f e c t e d . Aerosol Exposure of Mice Figure 10 and Table 8 i l l u s t r a t e the l e v e l s of v i r u s content i n various mouse t i s s u e s a f t e r aerosol i n o c u l a t i o n . Each value shown i s the mean of f i v e separate experiments f o r each t i s s u e examined. Values were w i t h i n two sigma u n i t s . I t should be noted that there was a two-day l a g before an increase i n the l e v e l of Powassan v i r u s i n the nasal e p i t h e l i u m was observed. This i s i n c o n t r a s t to an immediate increase a f t e r i n t r a n a s a l i n s t i l l a t i o n o f the v i r u s . Increase of v i r u s w i t h i n the lungs began immediately a f t e r aerosol i n o c u l a t i o n . I t rose from undetectable l e v e l s i n i t i a l l y to more than 5 log by the f i f t h day a f t e r aerosol i n o c u l a t i o n . A 24 hour l a g p e r i o d took place before appreciable viremia was 50 observed. At t h i s point approximately 2- to 3-log of v i r u s were present i n the lung. A f t e r l e v e l s i n the blood had r i s e n s i g n i f i c a n t l y v i r u s began to appear i n other t i s s u e s . Levels of v i r u s i n the spleen lagged behind, but c l o s e l y p a r a l l e l e d l e v e l s o f v i r u s w i t h i n the blood. I f they appear s l i g h t l y higher i t i s because o f c h a r a c t e r i s t i c p o o l i n g of blood with the t i s s u e s o f the spleen. During the course of t h i s i n v e s t i g a t i o n , various a l i q u o t s of blood were examined f o r blood eel 1-associated viremia as compared to plasma-a s s o c i a t e d v i r e m i a . I t was found that Powassan viremia was e s s e n t i a l l y plasma-borne and not red or white c e l l - a s s o c i a t e d (Mims, 1964). Blood c e l l s were separated from plasma, ground i n d i l u e n t and inocu-l a t e d i n t o mice to provide information whether the v i r u s was red or white c e l l -a s s o c i a t e d or was contained s o l e l y i n the plasma p o r t i o n o f the blood. Levels of v i r u s w i t h i n l i v e r t i s s u e were low; the time of increase approximated the r i s e of v i r u s l e v e l s i n the blood. Levels of Powassan v i r u s i n thymus, kidney, s k e l e t a l , c a r d i a c and smooth muscle t i s s u e s t r o n g l y r e f l e c t e d the trends o f v i r e m i a . M u l t i p l i c a t i o n of v i r u s w i t h i n the b r a i n was f i r s t detected three days a f t e r aerosol i n h a l a t i o n . This was one day a f t e r viremia became manifest. Levels of v i r u s w i t h i n the b r a i n f a r exceeded l e v e l s i n a l l other t i s s u e s , and a t t a i n e d about 9 lo g f i v e days or more a f t e r aerosol i n o c u l a t i o n . Histopathology Thin s e c t i o n s of various t i s s u e s were s t a i n e d with H and E f o r use with l i g h t microscope techniques, FITC, conjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n f o r use with fluorescence microscope techniques and osmium t e t r o x i d e followed by p o s t - s t a i n i n g with uranyl acetate and lead c i t r a t e f o r use with e l e c t r o n microscope techniques. 51 P l a t e 6 shows an H and E s t a i n e d s e c t i o n of an uninfected mouse lung. Plates 7 and 8 show H and E s t a i n e d s e c t i o n s of mouse lungs f i v e days a f t e r aerosol i n o c u l a t i o n with Powassan v i r u s . Plates 6 and 7 are magnified 160 x; P l a t e 8, 320 x. In i n f e c t e d lung, a l v e o l a r walls were swollen with accumulation o f macrophages and c a p i l l a r i e s were d i l a t e d (Plates 7 and 8). The walls were t h i -ckened i n c o n t r a s t to uninfected lung ( P l a t e 6). Thus i n f e c t e d lung showed patches of bronchopneumonic c o n s o l i d a t i o n . Within the cerebellum, v i r u s i n f e c t e d t i s s u e s showed i n f i l t r a t i o n with macrophages, e s p e c i a l l y surrounding degenerating neurones (Plates 10, 11) i n c o n t r a d i s t i n c t i o n to the lack o f i n f i l t r a t i o n i n uninfected t i s s u e s ( P l a t e 9). Within the cerebrum (Plates 12, 13, 14) widespread neuronal degenera-t i o n accompanied by macrophage accumulation was observed i n s u b c o r t i c a l areas and adjacent to cleavage planes. Plates 15 and 16 are c r o s s - s e c t i o n s of Powassan v i r u s i n f e c t e d mouse lung s t a i n e d with f l u o r e s c e n t - a n t i b o d y three and f i v e days a f t e r aerosol inocu-l a t i o n . This was an important f i n d i n g and i s probably the manner i n which Powassan v i r u s invades the blood stream. Plates 17 to 20 are f l u o r e s c e n t - a n t i b o d y s t a i n e d s e c t i o n s of Powassan vi r u s i n f e c t e d mouse br a i n three days a f t e r aerosol i n o c u l a t i o n showing examples of the cuboidal e p i t h e l i u m o f the choroid plexus. These c e l l s show Powassan vi r u s s p e c i f i c fluorescence and probably represent the means by which Powassan vi r u s crosses the blood-brain b a r r i e r to enter the cerebrospinal f l u i d and thus promote i t s dissemination through the c e n t r a l nervous system. P l a t e 21 shows Powassan v i r u s s p e c i f i c fluorescence i n cerebral cortex c e l l s adjacent to blood vessels i n the arachnoid l a y e r o f the meninges j u s t beneath the s k u l l three days a f t e r aerosol i n o c u l a t i o n . However, no immu-nofluorescence was demonstrated i n the o l f a c t o r y bulbs or t r a c t s before i t was observed elsewhere i n the b r a i n . 52 P l a t e 22 i s a s a g i t t a l s e c t i o n of i n f e c t e d mouse b r a i n showing widespread Powassan v i r u s s p e c i f i c fluorescence i n c e l l s o f the cerebral cortex s i x days a f t e r aerosol i n o c u l a t i o n . P l a t e 23 i s an e l e c t r o n micrograph o f what i s b e l i e v e d to be a c e l l of the r e t i c u l o e n d o t h e l i a l system i n the mouse lung three days a f t e r aerosol i n o c u l a t i o n with Powassan v i r u s . A t t e n t i o n i s c a l l e d to s t r u c t u r e s which resemble phagolysosomes s t r u c t u r e s known to be a c t i v e i n phagocytic c e l l s i n the presence o f f o r e i g n m a t e r i a l . Plates 24, 25, and 26 are e l e c t r o n micrographs o f nasal e p i t h e l i u m three days a f t e r i n t r a n a s a l i n s t i l l a t i o n of Powassan v i r u s . Virus s p e c i f i c s t r u c t u r e s were observed i n i n f e c t e d e p i t h e l i u m but never i n the uninfected e p i t h e l i u m that was examined. DISCUSSION The pathogenecity of Powassan v i r u s f o r the s u c k l i n g mouse was always more r a p i d and intense than i n the a d u l t . Virus t i t r e s reached a peak i n s i x days i n the s u c k l i n g mouse, compared to e i g h t days i n the a d u l t . The hind limbs o f i n f e c t e d mice with rare exception became paralyzed. The p a r a l y s i s ascended to the t h o r a c i c region of the mouse which induced a 'hunched back' appearance before death. Midway i n the i n f e c t i o n mice were hy p e r e s t h e t i c to l i g h t , sound and touch. This slowly diminished u n t i l the i n f e c t e d mouse became i n s e n s i t i v e to several s t i m u l i . The above sequence was followed by coma and death. Because a r e l i a b l e t i s s u e c u l t u r e plaque assay method (Cooper, 1967) f o r Powassan v i r u s has not as y e t been developed, the assessment o f the v i r u s content o f Powassan v i r u s suspensions was based on the sigmoid dose-response curve. Since an absolute dosage (one that w i l l j u s t a f f e c t a l l e n t i t i e s tested) cannot be measured, the 50 p e r c e n t i l e , or the dosage 53 causing an e f f e c t ( i n the case o f Powassan virus, l e t h a l i t y i n mice) i n 50 per cent o f e n t i t i e s t e s t e d was used. These data were analyzed by the s t a t i s t i c a l method o f Reed and Muench (1938). Brown (1964) i n a paper on variance e s t i m a t i o n using the Reed and Muench 50 per cent end p o i n t determination s t a t e d that i f the number o f s u s c e p t i b l e animals used f o r the determination was constant, and greater than f i v e f o r each d i l u t i o n examined f o r v i r u s content, the two sigma l i m i t s com-puted are comparable with those obtained using other procedures i n which e s t i -mates o f sample v a r i a t i o n accompany the procedure ( B l i s s , 1938), (Wilson and Worcester, 1943), ( M i l l e r and T a i n t e r , 1944), (Finney, 1947), ( L i t c h f i e l d and Wilcoxan, 1949), (Finney, 1952). Nairn (1969) recommended negative pressure d i a l y s i s f o r the con-c e n t r a t i o n and p u r i f i c a t i o n o f FITC conjugated gamma g l o b u l i n a f t e r G-25 Sephadex f i l t r a t i o n . It was found during t h i s study t h a t D i a f l o u l t r a f i l t r a -t i o n techniques using XM50 membranes ( r e t a i n s a l l molecules with a molecular weight greater than 50,000) were s u p e r i o r f o r the p u r i f i c a t i o n and concentra-t i o n o f conjugated g l o b u l i n s . No chance rupture o f d i a l y s i s tubing and the consequent l o s s o f sample can occur using the D i a f l o technique. L y o p h i l i z e d u n infected s u c k l i n g mouse brain was used f o r adsorbing non-virus antibody from FITC conjugated gamma g l o b u l i n . L i v e r preparations were found g e n e r a l l y to contain unacceptably high concentrations o f hemo-gl o b i n . The hemoglobin tended to mask fluorescence on fl u o r e s c e n t - a n t i b o d y t r e a t e d t i s s u e s e c t i o n s . Determination o f the minimum l e t h a l dose by various routes o f i n o c u l a t i o n revealed that the i n t r a c e r e b r a l and intravenous routes require the same dose. Mims (1960), who c a r r i e d out experiments on i n t r a c e r e b r a l i n j e c t i o n s i n mice, s t a t e d that the pressures exerted during routine i n t r a -c e r e b r a l i n j e c t i o n s ranged from 200 to 300 cm. of water o r 20 to 30 times 54 normal cerebrospinal f l u i d pressure. I t would not be s u r p r i s i n g i f anato-mical b a r r i e r s were broken under such high i n j e c t i o n pressures. Mims be l i e v e d that the f i r s t b a r r i e r s to break down as the pressure rose were the arachnoid v i l l i . The l a r g e endocranial venous sinuses are e n t i r e l y enclosed by t h i c k w alls o f dura matter except i n d e f i n i t e p l a c e s , c h i e f l y i n the s a g i t -t a l sinus o f the f a l x (the process o f dura which intervenes between the cere-bra l hemispheres) where the dura i s p e r f o r a t e d by numerous p r o t r u s i o n s of the arachnoid membrane, through each of which a f i n g e r - l i k e evagination o f the arachnoid mesothelium i s t h r u s t i n t o the lumen o f the s i n u s . This i s an archnoid v i l l u s . Its c a v i t y , which contains a small amount o f loose arach-noid t i s s u e i s i n f r e e communication with the subarachnoid spaces, so that here the f l u i d o f these spaces i s separated from the blood o f the sinus only by a t h i n mesothelial membrane. The arachnoid v i l l i provide the main pathway f o r the outflow o f cerebrospinal f l u i d d i r e c t l y i n t o the venous c i r c u l a t i o n ; once they have been 'blown open' there i s a f r e e flow o f i n j e c t e d material i n t o the blood stream and from t h i s p o i n t on the i n j e c t i o n becomes an intravenous one. The d i f f e r e n c e s i n l e t h a l dose that were observed between the sub-cutaneous and the i n t r a p e r i t o n e a l routes o f i n o c u l a t i o n can probably be a s c r i b e d to the d i f f e r e n c e s i n s t r u c t u r e , phagocytic index, m o t i l i t y , enzyme content o f lysosomes, oxygen requirements and other metabolic and b i o -chemical c h a r a c t e r i s t i c s o f macrophages i n the subcutaneous t i s s u e s as com-pared to the p e r i t o n e a l c a v i t y of the mouse (Johnson, 1964), (Fenner, 1968), Nelson, 1969). As mentioned mice could not be i n f e c t e d by the i n s t i l l a t i o n o f high concentrations o f Powassan v i r u s i n t o the stomach. This event i s i n strong c o n t r a s t to the f a c t that a r e l a t e d v i r u s tick-borne e n c e p h a l i t i s v i r u s can 55 cause a f a t a l i n f e c t i o n i n mice when introduced i n t o the g a s t r o i n t e s t i n a l t r a c t (Pogodina, 1962), (Moritsch and Kovack, 1962). Tick-borne encepha-l i t i s v i r u s penetrates and r e p l i c a t e s i n the i n t e s t i n e followed by viremia with i n v a s i o n and m u l t i p l i c a t i o n i n the c e n t r a l nervous system. In the case of Powassan v i r u s the gut c e l l s e i t h e r do not possess receptor s i t e s f o r the v i r u s or i t becomes i n a c t i v a t e d by stomach acids or the a c t i v e i n g r e d i e n t s o f b i l e (sodium deoxycholate) ( T h e i l e r , 1957) w i t h i n the i n t e s t i n e . When mice were subjected to Powassan v i r u s i n f e c t i o n by the i n t r a -nasal i n s t i l l a t i o n of v i r u s they contracted a f u l m i n a t i n g i n f e c t i o n and died w i t h i n e i g h t days. Because l a t e r s t u d i e s i n d i c a t e d that mice could not be i n f e c t e d by the i n s t i l l a t i o n of l a r g e concentrations of Powassan v i r u s d i r e c t l y i n t o the lumen o f the g a s t r o i n t e s t i n a l t r a c t i t must be assumed t h a t the presence o f v i r u s i n the f l u i d which bathed the nasopharynx and entered the lung caused the i n f e c t i o n . The f a c t t h a t v i r u s c o n t a i n i n g f l u i d entered the lung i s pro-ven by the presence at zero time o f approximately 3 log o f v i r u s . Subsequen-t l y , the Powassan v i r u s t i t r e i n the lung t i s s u e exceeded 6 l o g . A drop o f v i r u s - c o n t a i n i n g f l u i d was placed on the external nares of l i g h t l y anaesthetized mice. Upon breathing the f l u i d was a s p i r a t e d d i r e c t l y i n t o the lungs; a l s o the nasopharynx was bathed i n the i n f e c t i o u s v i r u s suspension. Nasal e p i t h e l i u m became i n f e c t e d and the important obser-vatio n was that the v i r u s began to m u l t i p l y immediately with no i n t e r v e n i n g l a g p e r i o d . Viremia became measurable a f t e r a l a g p e r i o d o f from one to two days a f t e r i n o c u l a t i o n . The i n f e c t e d t i s s u e s c o n t r i b u t i n g to t h i s viremia were with l i t t l e doubt the e p i t h e l i a l t i s s u e s o f the nasopharynx and lungs. A f t e r a s i m i l a r l a g p e r i o d v i r u s appeared i n the c e n t r a l nervous system and 5 6 m u l t i p l i e d to a high l e v e l . Virus l e v e l s i n the spleen r e f l e c t the time sequence o f viremia and are s l i g h t l y higher because o f the c h a r a c t e r i s t i c p o o l i n g o f blood w i t h i n s p l e n i c t i s s u e . The apparently large v i r u s dose req u i r e d to i n i t i a t e i n f e c t i o n and cause death i n mice i n o c u l a t e d by the i n t r a n a s a l i n s t i l l a t i o n o f Powassan v i r u s was probably due to the a b i l i t y o f the c i l i a t e d e p i t h e l i u m o f the tracheobron-c h i a l tree to remove a considerable q u a n t i t y o f the inoculum to the region o f the oropharynx where i t could be expectorated o r swallowed. In the stomach o f course i t was shown that the v i r u s i n the inoculum was i n a c t i v a t e d . The absence of immunofluorescence i n the o l f a c t o r y bulb neurones before i t was demonstrated elsewhere i n the b r a i n suggests that i n f e c t i o n o f the c e n t r a l nervous system d i d not r e s u l t from entry o f v i r u s through the o l f a c t o r y p l a t e . The mechanism o f upper r e s p i r a t o r y t r a c t clearance ( e x c l u s i v e o f nasal passages) i s dependent upon proximal movement o f a blanket o f mucinous f l u i d which i s maintained by the c i l i a r y a c t i v i t y o f columnar e p i t h e l i u m l i n i n g the tracheobronchial mucus membrane. This c i l i a t e d e p i t h e l i u m extends from the i n f e r i o r aspect of the e p i g l o t t i s down to the terminal b r o n c h i o l e . Not a l l these c e l l s , o f course are c i l i a t e d . In the r a t the r a t i o o f n o n - c i l i a t e d (goblet c e l l s ) to c i l i a t e d c e l l s i s 3 : 5 . The beat frequency of the c i l i a i s about 1 3 0 0 per minute. This a c t i v i t y r e s u l t s i n the propulsion o f the o v e r y l i n g mucinous f l u i d a t an average rate of approximately 1 3 . 5 mm. per minute (Hatch and Gross, 1 9 6 4 ) . The c a l c u l a t e d l e t h a l dose 5 0 per cent by the aerosol route o f i n o c u l a t i o n was 6 4 0 M I C L D ^ Q. This i s probably an over-estimate o f the actual r e t a i n e d dose o f Powassan v i r u s when mice were subjected to i n f e c t i o u s a e r o s o l s . The 6 4 0 M I C L D 5 Q assumes t h a t a l l the aerosol that was inhaled by the mouse i n a pr e s c r i b e d time was re t a i n e d . I t should be noted that an experimental animal both i n h a l e s and swallows microbes as a r e s u l t o f res-p i r a t o r y exporuse. Goldberg and L e i f ( 1 9 5 0 ) exposed mice to a t e s t aerosol 57 of P32-1abe11ed P a s t e u r e l l a p e s t i s and found that whereas the t o t a l r e t e n t i o n was over 80 per cent o f the c a l c u l a t e d dose per mouse only 30 per cent o f the re t a i n e d material ( i . e . , 24 per cent o f the t o t a l ) was i n the r e s p i r a t o r y tree and 70 per cent was i n the g a s t r o i n t e s t i n a l t r a c t . Between s p e c i e s , lung volume i s pr o p o r t i o n a l to body weight, whereas a l v e o l a r s u r f a c e area c o r r e l a t e s l i n e a r l y and d i r e c t l y with metabolic a c t i -v i t y (Tenney and Remmers, 1963). Two animals o f equal body s i z e w i l l have lungs o f the same volume, but i f one has a higher rate o f metabolism i t s a l v e o l i w i l l be smaller. P a r t i c l e s i z e i s t h e r e f o r e an extremely c r i t i c a l f a c t o r f o r r e s p i r a t o r y penetration and r e t e n t i o n i n l a b o r a t o r y animals. With a homogenous aerosol of 1 to 2 micron s i z e d p a r t i c l e s , average r e s p i r a t o r y r e t e n t i o n i s 15 per cent i n the mouse (Dimmick and Ackers, 1969), (Danes e_t a l , 1962). I f t h i s was a p p l i e d to the t h e o r e t i c a l LD^Q by an aerosol i t would amount to about 96 M I C L D ^ Q or approximately e q u i v a l e n t to the LD^Q by way o f i n t r a p e r i t o n e a l i n o c u l a t i o n . Because the c a l c u l a t e d LD^Q by aerosol i n o c u l a t i o n was determined to have been rec e i v e d a f t e r 22 seconds o f exposure to the a e r o s o l , the Powa-ssan v i r u s r e t e n t i o n curve appears to f a r underestimate the actual dose re-ta i n e d a f t e r f i v e minutes o f exposure to the a e r o s o l . It would o f course be impossible to recover the t o t a l number o f ret a i n e d M I C L D ^ Q from exposed lungs. During the process o f t i s s u e g r i n d i n g released enzymes would probably i n a c t i v a t e some o f the v i r u s , thermal and l i g h t i n a c t i v a t i o n would a l s o take i t s t o l l ; a l s o p o s s i b l y a la r g e proportion o f v i r u s would be adsorbed to di s r u p t e d lung t i s s u e and would be sedimented with unwanted c e l l u l a r debris during c e n t r i f u g a t i o n ( A l b r e c h t , 1962), (Appleyard, 1967). In some of the longer duration exposures much o f the v i r u s could have entered c e l l s to e x i s t i n the e c l i p s e phase o f r e p l i c a t i o n . 58 The curve does show, however, t h a t the longer the mouse i s held i n the aerosol the g r e a t e r the degree o f r e t e n t i o n . The dose-retention curve may more c l o s e l y approximate the actual happenings. With the r e c i p r o c a t i n g p a t t e r n o f a i r flow i n t o and out o f the r e s p i r a t o r y system, only a f r a c t i o n o f each t i d a l volume reaches the p u l -monary a i r spaces; the r e s t f i l l s the nasopharyngeal chamber and airways o f the upper r e s p i r a t o r y t r a c t . C l e a r l y no more aerosol can be c a r r i e d to the pulmonary a i r spaces than i s contained i n the f r a c t i o n o f new a i r . I t has a l s o been shown that only l i m i t e d mixing takes place from breath to breath and t h i s f a c t has i n t e r e s t i n g i m p l i c a t i o n s with respect to depth of penetra-t i o n i n t o and s i t e o f d e p o s i t i o n o f aerosol p a r t i c l e s i n the pulmonary a i r spaces. Since the bulk o f new a i r does not mix v o l u m e t r i c a l l y with lung a i r i t follows t h a t n o n - d i f f u s i b l e p a r t i c l e s (greater than 0.5 micron) w i l l penetrate only as f a r as the new a i r goes. Sub-micron ( i . e . , l e s s than 0.5 micron) having s i g n i f i c a n t d i f f u s i o n v e l o c i t i e s w i l l to some degree move independently i n t o the s t a t i c lung a i r j u s t as do gas molecules. There can be s i g n i f i c a n t d i f f e r e n c e s with aerosol p a r t i c l e s i z e in the s i t e o f d e p o s i t i o n w i t h i n the ultimate pulmonary u n i t . The coarser p a r t i c l e s w i l l be deposited i n g r e a t e r degree high up i n the u n i t (along r e s p i r a t o r y bronchioles and a l v e o l a r d u c t s ) ; whereas the sub-micron p a r t i c l e s w i l l have a r e l a t i v e l y g r e a t e r p r o b a b i l i t y of being deposited i n the alveo-l a r sacs and a l v e o l i (Hatch and Gross, 1964). Powassan v i r u s e i t h e r i n suspensions or i n aerosols proved to be h i g h l y s e n s i t i v e to i n a c t i v a t i o n by u l t r a v i o l e t l i g h t . Appleyard (1967) found that Semliki Forest v i r u s and to a l e s s e r extent Sindbis v i r u s , two group A v i r u s e s , Murray V a l l e y e n c e p h a l i t i s v i r u s , a group B v i r u s , i n f l u e n z a type A and r a b b i t pox were s e n s i t i v e to natural or a r t i f i c i a l d a y l i g h t . The a c t i v e wave lengths were i n the region of from 3300 to 4700 Angstrom u n i t s . 59 Kleczkowski (1957), Gard and Maaloe (1959), Kleczkowski (1968) described a c t i o n s p e c t r a as curves obtained by p l o t t i n g wave lengths o f r a d i a t i o n s a g a i n s t t h e i r r e l a t i v e e f f i c i e n c i e s i n causing i n a c t i v a t i o n . Coincidence between a maximum o f the a c t i o n spectrum and a maximum o f the absorption spectrum of some component of the v i r u s may be considered as an i n d i c a t i o n that the component might be concerned i n the mechanism o f i n a c t i v a t i o n . A c t i o n s p e c t r a o f v a c c i n i a v i r u s , o f a few bacteriophages and o f i n f l u e n z a A a l l resemble the absorption spectrum of n u c l e i c a c i d . I t could be concluded t h e r e f o r e that the n u c l e i c a c i d components o f the v i r u s e s are i n v o l v e d i n the mechanism o f i n a c t i v a t i o n o f these v i r u s e s by u l t r a v i o l e t i r r a d i a t i o n . I t was found that two peaks i n the a c t i o n spectrum of a Megatherium bacteriophage a broad and prominent one at around 2600 Angstrom uni t s cor-responding with the maximum adsorption f o r n u c l e i c a c i d s , and a s m a l l e r one at 2800 Angstrom units corresponding with t y r o s i n e and tryptophan residues o f p r o t e i n . There was also a sharp r i s e o f the a c t i o n spectrum curve as the wave length f e l l below 2300 Angstrom units where adsorption by a l i p h a t i c amino acids and the peptide bond became pronounced. I t was a l s o revealed that a wave length o f 2652 Angstrom uni t s was the most e f f i c i e n t i n i n a c t i v a t i n g the a b i l i t y of i n f l u e n z a v i r u s to i n f e c t and m u l t i p l y , whereas that of 2803 Angstrom units was the most e f f i c i e n t i n i n a c t i v a t i n g i t s hemagglutinating a b i l i t y . The n u c l e i c a c i d component of the v i r u s therefore may be p r i m a r i l y concerned with l o s s o f a b i l i t y to i n f e c t and m u l t i p l y , whereas the p r o t e i n component may be concerned with l o s s of the a b i l i t y to hemagglutinate. Powassan v i r u s aerosols were s l i g h t l y more s t a b l e at low r e l a t i v e humidity than at intermediate and/or high r e l a t i v e h u m i d i t i e s . S i m i l a r obser-vations were made when Semliki Forest v i r u s aerosols were examined f o r b i o -l o g i c a l decay with time (Gaunt, 1968, 1969). Semliki Forest v i r u s , how-ever, was s l i g h t l y more s e n s i t i v e to b i o l o g i c a l decay at 80 per cent r e l a t i v e humidity than a t 50 per cent r e l a t i v e humidity which was demon-s t r a t e d with Powassan v i r u s . Harper (1961) examining the airborne s u r v i v a l o f v a c c i n i a , i n f l u -enza, Venezuelan equine encephalomyelitis and p o l i o m y e l i t i s v i r u s e s , found b e t t e r s u r v i v a l a t low r e l a t i v e humidities.for v a c c i n i a , i n f l u e n z a and Vene-zuelan equine e n c e p h a l o m y e l i t i s ; on the other hand* p o l i o v i r u s s u r v i v e d b e t t e r a t high r e l a t i v e humidity. Webb e t al_, (1963) s t u d i e d the e f f e c t o f r e l a t i v e humidity on aerosols o f pigeon pox v i r u s and Rous Sarcoma v i r u s i n the presence and absence o f i n o s i t o l . , Pigeon pox v i r u s was found to be s t a b l e i n aerosols and was l i t t l e a f f e c t e d by changes i n r e l a t i v e humidity. Rous Sarcoma v i r u s however, was extremely s e n s i t i v e to r e l a t i v e humidity. Webb has suggested t h a t the death o f airborne b a c t e r i a i s a d i r e c t r e s u l t o f l o s s o f water molecules bound to c e l l nucleoproteins and that the a c t i o n o f p r o t e c t i v e compounds, such as i n o s i t o l , r e s u l t s from t h e i r a b i l i t y to take over the r o l e o f bound water i n maintaining the v i t a l s t r u c t u r e o f these c e l l com-ponents. When Rous Sarcoma v i r u s was a e r o s o l i z e d from a s i x per cent s o l u -t i o n o f i - i n o s i t o l the recovery o f v i r u s at 30 per cent r e l a t i v e humidity increased from 1 per cent to 90 per cent. This p r o t e c t i v e a b i l i t y o f ino-s i t o l was apparent at a l l r e l a t i v e humidity values below 70 per cent (values above which Rous Sarcoma v i r u s was most s t a b l e ) . Dejong and Winkler (1968) working with aerosols o f p o l i o v i r u s s t a t e d t hat an organism sprayed i n a i r i s exposed to (1) the s t r e s s o f spr a y i n g , (2) quick evaporation u n t i l the dr o p l e t s are i n e q u i l i b r i u m with with ambient a i r , and (3) to the decay i n the s t a b l e aerosol during s t o r -age. They a l s o s a i d t h a t the mechanisms of i n a c t i v a t i o n during these three 61 phases might d i f f e r . T h e i r data revealed t h a t the spray l o s s was l a r g e a t low r e l a t i v e humidity and decreased with r i s i n g humidity, being minimum at 55 per cent r e l a t i v e humidity. The storage l o s s rate was low below 35 per cent r e l a t i v e humidity and above 70 per cent r e l a t i v e humidity, but high between 40 and 55 per cent r e l a t i v e humidity. The range o f maximal storage loss rate co-i n c i d e s with the range o f decrease o f the spray l o s s . In order to determine the rate o f o x i d a t i o n , p o l i o v i r u s was aero-s o l i z e d i n the presence of nitrogen and in the presence o f oxygen. As well as f o l l o w i n g the b i o l o g i c a l decay o f the i n t a c t p o l i o v i r u s p a r t i c l e , i n f e c -t i o u s r i b o n u c l e i c a c i d was also e x t r a c t e d from samples o f v i r u s taken a t d i f f e r e n t time i n t e r v a l s during aerosol storage. The observations o f Dejong and Winkler were t h a t : (1) the r i b o -n u c l e i c a c i d i n the v i r u s decays simultaneously with the whole v i r u s p a r t i c l e , (2) o x i d a t i o n does not play a s i g n i f i c a n t r o l e , (3) L- c y s t i n e which s t a b i l i z e s aqueous s o l u t i o n s o f p o l i o v i r u s a g a i n s t thermal i n a c t i v a t i o n at 50°C by r e a c t i n g with SH-groups and s t a b i l i z i n g s t r u c t u r e , does not p r o t e c t p o l i o v i r u s aerosols from decay. On the strength o f these observations i t was concluded that i n aerosols denaturation o f v i r a l r i b o n u c l e i c a c i d i s the cause o f the i n a c t i v a -t i o n o f p o l i o v i r u s . Like Powassan v i r u s , Benbough (1971) showed that Langat v i r u s , a member o f the group B tick-borne a r b o v i r u s e s , d i d not s u r v i v e as well i n aerosols held a t low, intermediate and high r e l a t i v e humidites when the spray f l u i d contained s a l t s . He l a t e r found s u r v i v a l was ap p r e c i a b l y b e t t e r f o r Langat v i r u s when the v i r u s was nebulized from d e s a l t e d s o l u t i o n s . 62 Benbough b e l i e v e s that the s u s c e p t i b i l i t y o f vi r u s e s i n aerosols to the environmental conditions must be r e l a t e d to the p a r t i t i o n o f bound and unbound water between v i r u s , other c o n s t i t u e n t s o f the aerosol p a r t i c l e c o n t a i n i n g the v i r u s , and the surrounding atmosphere. He maintained that f o l l o w i n g a e r o s o l i z a t i o n there i s a p e r i o d o f e q u i l i b r a t i o n during which the concentration o f so l u t e s i n the aerosol p a r t i c l e increases to l e v e l s which may be t o x i c to the v i r u s . Benbough noted l i t t l e e f f e c t on the b i o l o g i c a l decay of p o l i o v i r u s regardless o f whether the v i r u s was a e r o s o l i z e d from s a l t e d or desa l t e d s o l u t i o n s . He p o s t u l a t e d that the i n a c t i v a t i o n o f arboviruses n e b u l i z e d from f l u i d s c o n t a i n i n g s a l t s could be due to the d i s s o l u t i o n o f the l i p o -p r o t e i n s which are present i n the v i r u s . This breakdown of l i p o p r o t e i n s o f viruses i n aerosols may be caused by c h l o r i d e ions d i s p l a c i n g bound water i n membrane systems and that arboviruses having v a r i a b l e amounts o f l i p o -p r o t e i n content could be expected to have d i f f e r e n t s u s c e p t i b i l i t i e s to s a l t s i n a e r o s o l s . My i n t e r e s t was i n determining the decay of Powassan v i r u s i n the types o f f l u i d suspensions i n which the v i r u s i s o r d i n a r i l y handled and i n the l i f e o f the v i r u s when aerosols e i t h e r a c c i d e n t a l l y or i n t e n t i o n a l l y were created from such suspensions. In the c r o s s - i n f e c t i o n study aerosol exposed mice were placed i n a separate cage f o r 24 hours so that they could complete c h a r a c t e r i s t i c grooming p r a c t i c e s which were observed to occur a f t e r f u l l body exposure o f mice to i n f e c t i o u s aerosols o f Powassan v i r u s . The grooming, both i n d i -v i d u a l l y and c o l l e c t i v e l y i n v o l v e d the l i c k i n g o f f u r plus the l i c k i n g o f f r o n t f e e t i n combination with a washing a c t i o n . These grooming p r a c t i c e s sometimes continued f o r several hours a f t e r aerosol exposure. I t i s an educated guess that any v i r u s adhering to f u r would be removed by these 63 p r a c t i c e s or would become i n a c t i v a t e d n a t u r a l l y w i t h i n 24 hours. A f t e r 24 hours uninfected mice and i n f e c t e d mice were placed to-gether i n a new, clean cage which was fed with a f i l t e r e d , h u m i d i f i e d a i r supply. Mice were maintained f o r 30 days. This l i m i t e d study i n d i c a t e d t h a t uninfected mice can become c r o s s -i n f e c t e d by mice who have been exposed and become i n f e c t e d by aerosols o f Powassan v i r u s . The p o s s i b i l i t y i s f a i r l y high that mice became i n f e c t e d by nose-to-nose contact; that i s the a c c i d e n t a l i n h a l a t i o n by an uninfected mouse o f p a r t i c l e s produced by an aerosol i n f e c t e d mouse upon s n u f f l i n g or sneezing. The other p o s s i b i l i t i e s are that mice become i n f e c t e d by the i n h a l a t i o n of aerosols o f urine or f e c e s . This i s not as l i k e l y because mice were removed r e g u l a r l y to clean cages with care being e x e r c i s e d so that a c c i d e n t a l aerosols with regard to feces or urine were not created. Also any urine was soon adsorbed by clean new shavings and i t was noted that feces from mice seldom became powdery (because o f c o n t r o l l e d humidity), a s i t u a t i o n which would be conducive to the c r e a t i o n o f i n f e c t i o u s dusts. Aerosol i n f e c t e d mice succumbed e i g h t to ten days a f t e r exposure. C r o s s - i n f e c t e d mice succumbed 10 to 14 days a f t e r t h a t ; i n d i c a t i n g p o s s i b l y that uninfected mice re c e i v e d r e l a t i v e l y small doses o f v i r u s from t h e i r aerosol i n f e c t e d counterparts which e v e n t u a l l y m u l t i p l i e d to s i g n i f i c a n t l e v e l s i n the lungs and subsequently caused t h e i r deaths 10 to 14 days l a t e r . These experiments provide a model of a s i t u a t i o n whereby vir u s may be t r a n s f e r r e d to a c l o s e human contact by a person who has become i n f e c t e d a c c i -d e n t a l l y by i n h a l a t i o n o f an a e r o s o l . Druett e t al_ (1956) found that there was a p o s i t i v e c o r r e l a t i o n between the number o f deaths i n control animals and the number o f dead or dying experimentally i n f e c t e d animals with which the c o n t r o l s were i n con-t a c t . The s i z e o f p a r t i c l e s i n f e c t i n g experimental animals a l s o i n f l u e n c e d the c r o s s - i n f e c t i o n r a t e . Deaths i n control animals were approximately four times g r e a t e r a f t e r contact with animals exposed to p a r t i c l e s c o n t a i n i n g 64 only one c e l l than a f t e r contact with animals p r e v i o u s l y exposed to m u l t i -c e l l u l a r p a r t i c l e s o f 12 microns i n diameter. The above of course can be c o r r e l a t e d with d e p o s i t i o n o f large aerosol p a r t i c l e s i n the upper r e s p i r a t o r y t r a c t and clearance by mucoci-l i a r y mechanism. I t was n o t i c e d a f t e r exposure o f mice to aerosols o f Powassan vir u s that growth w i t h i n the turbinates lagged, f o r from two to three days before a no t i c e a b l e i n c r e a s e i n v i r u s was observed. At t h i s p o i n t i n time l e v e l s i n the blood were between 3 and 4 l o g . This suggests that the i n f e c t i o n o f the nasal e p i t h e l i u m was a r e s u l t o f viremia rather than d i r e c t contact between nasal e p i t h e l i u m and the i n f e c t i o u s a e r o s o l . S i m i l a r l y , the c e n t r a l nervous system was not i n f e c t e d by t r a n s f e r o f v i r u s through the o l f a c t o r y p l a t e s . The C o l l i son atomizer produces p a r t i c l e s i n the 1 to 5 micron s i z e range. P a r t i c l e s o f t h i s s i z e penetrate deeply i n t o the lung. P a r t i c l e s i n the 10 micron range and above are u s u a l l y trapped i n the nasopharynx (Hatch and Gross, 1964). When v i r u s was i n s t i l l e d i n t o the external nares growth i n the nasal e p i t h e l i u m began immediately. This i n d i c a t e s that under the condit i o n s o f nasal i n s t i l l a t i o n o f v i r u s , the ep i t h e l i u m which was bathed i n the inoculum became i n f e c t e d d i r e c t l y r a t h e r than by way of viremic blood. M u l t i p l i c a t i o n o f v i r u s w i t h i n the lung a f t e r aerosol i n o c u l a t i o n began immediately and l e v e l s rose from undetectable amounts to greater than 5 l o g i n f i v e days. This was i n co n t r a s t to mice which were i n f e c t e d by the i n t r a n a s a l i n s t i l l a t i o n o f v i r u s where 3 log was recovered d i r e c t l y from the lung t i s s u e immediately a f t e r i n o c u l a t i o n . The r i s e o f v i r u s i n the thymus t i s s u e may be as a r e s u l t o f m u l t i -p l i c a t i o n o f v i r u s w i t h i n t h i s t i s s u e or w i t h i n many o f the t h o r a c i c lymph nodes which are c l o s e l y a s s o c i a t e d with the thymus gland and which were pro-bably removed with the gland when i t was separated from t i s s u e s o f the lungs, 65 heart and l a r g e v e s s e l s . A l t e r n a t i v e l y the v i r u s t i t r e may simply r e f l e c t the concentration o f v i r u s i n the blood which was incompletely removed during p r o c e s s i n g of t i s s u e . Bloom and Fawcett (1968) re p o r t that i n the thymic r e t i c u l u m , macro-phages are l o c a t e d e s p e c i a l l y i n the v i c i n i t y o f blood v e s s e l s . These macro-phages could engulf v i r u s p a r t i c l e s and i n i t i a t e an i n f e c t i o n i n thymus t i s s u e . The blood supply to the thymus ( i n man) i s from a r t e r i e s which a r i s e from the i n t e r n a l mammary and the i n f e r i o r t h y r o i d veins. The lymphatics run mainly i n i n t e r ! o c u l a r connective t i s s u e and empty i n t o a n t e r i o r mediastinal and t r a c h i o -bronchial lymph nodes (nodes c l o s e l y a s s o c i a t e d with thymus t i s s u e ) . Blood supply and drainage i n the mouse would be comparable. I n f e c t i o u s blood would soon reach thymus t i s s u e from the s i t e o f i n i t i a l m u t l i p l i c a t i o n — the lung. Because the thymus gland i s i n t i m a t e l y i n v o l v e d i n the development o f immuno-l o g i c a l competence i n young mice (Blood and Fawcett, 1968) contact with b a c t e r i a and v i r u s e s would seem to be a n e c e s s i t y ; t h e r e f o r e i t i s not unrea-sonable that the t i s s u e s of the thymus gland and lymph nodes became i n f e c t e d with Powassan vir u s and was instrumental i n keeping l e v e l s of v i r u s w i t h i n the blood high. Levels of Powassan v i r u s i n the l i v e r were low. I t i s known ( T h i e l e r , 1957), ( A l b r e c h t , 1962) that arboviruses are h i g h l y s u s c e p t i b l e to b i l e s a l t s (sodium deoxycholate) or p o s s i b l y d e s t r u c t i v e enzymes released during t i s s u e g r i n d i n g when e x t r a c t i n g Powassan v i r u s from t i s s u e . The g a s t r o i n t e s t i n a l t r a c t could not be i n f e c t e d by i n s t i l l a t i o n of Powassan v i r u s d i r e c t l y . A f t e r aerosol i n f e c t i o n of mice the l e v e l of Powassan v i r u s i n gut t i s s u e was followed; minimal l e v e l s were detected s o l e l y on the f i f t h day. R i s i n g l e v e l s of v i r u s i n the kidney t i s s u e could mean that m u l t i -p l i c a t i o n took place i n t h i s organ. The increase c l o s e l y follows the time sequence f o r r i s i n g l e v e l s i n nasal e p i t h e l i u m and thymus gland t i s s u e . 66 Levels o f v i r u s i n s t r i a t e d muscle and c a r d i a c muscle r e f l e c t v i remia. Total removal o f viremic blood from these t i s s u e s would be impos-s i b l e by the methods that were used ( f i n e d i c i n g with s u c c e s s i v e washings). A more complete removal o f blood from various t i s s u e s can be accomplished by p e r f u s i o n o f the whole animal p r i o r to removal and processing o f i n f e c -ted t i s s u e s . Determinations o f hemoglobin i n t i s s u e s a f t e r whole body per-f u s i o n experiments have been as low as 10 per cent o f the o r i g i n a l hemo-gl o b i n and as high as 50 per cent ( A l b r e c h t , 1962). Of i n t e r e s t i s the increase o f l e v e l s o f v i r u s i n smooth muscle. The r e p r e s e n t a t i v e t i s s u e used f o r smooth muscle was the u r i n a r y bladder of the mouse. Levels o f v i r u s i n t h i s t i s s u e could stem from m u l t i p l i c a t i o n i n the smooth muscle t i s s u e o f the u r i n a r y bladder or the e p i t h e l i a l l a y e r o f c e l l s forming the mucosal s u r f a c e o f the bladder. The submucosa, the l a y e r o f connective t i s s u e interposed between the lamina p r o p r i a o f the mucosa and the outer band o f smooth muscle making up the bladder i s h i g h l y i n t e r s p e r s e d with blood vessels (Bloom and Fawcett, 1968). Viremic blood could i n i t i a t e an i n f e c t i o n i n t h i s t i s s u e . A l t e r n a t i v e l y , i n f e c t i o n of the e p i t h e l i a l c e l l o f the kidney could have taken place by the passage o f viremic blood through the glomeruli of the kidney. The capsule o f Bowman develops around a t u f t o f glomerular c a p i l l i a r i e s as a double walled cup composed o f squamous e p i t h e l i u m . The wall c l o s e l a p p l i e d to the glomerulus i s the v i s c e r a l l a y e r (glomerular epithelium) the outer w a l l , the p a r i e t a l l a y e r ( c a p s u l a r epithelium) and the s l i t - l i k e c a v i t y between them i s the capsular space (Bowman's space). At the vascular pole o f the renal c o r p u s c l e , the v i s c e r a l l a y e r i s r e f l e c t e d o f f the glomerular v e s s e l s to become continuous with the squamous e p i t h e l i u m of the p a r i e t a l l a y e r . At the u r i n a r y p o l e , the capsular e p i t h e -lium i s continuous with the cuboidal e p i t h e l i u m i n the neck o f the proximal 67 convoluted tubule. The v i s c e r a l l a y e r becomes e x t e n s i v e l y modified; i t bears f i l t r a -t i o n s l i t s about 250 Angstrom units wide, while glomerular endothelium i s pe r f o r a t e d by pores 500 to 1000 Angstrom units i n diameter (Bloom and Fawcett, 1968). It i s understandable that kidney e p i t h e l i u m would be r e l a t i v e l y easy to i n f e c t under these c o n d i t i o n s from an anatomical basis alone. I t i s known also that Powassan v i r u s w i l l adsorb to and m u l t i p l y w i t h i n a v a r i e t y o f renal e p i t h e l i a l t i s s u e (Abdelwahab e t al_, 1964), ( T a y l o r , 1967). I t i s als o reasonable that a f t e r v i r u s has m u l t i p l i e d w i t h i n the renal e p i t h e l i u m upon re l e a s e from i n f e c t e d c e l l s v i r u s could be c a r r i e d by the ureters to the uri n a r y bladder, the mucosal surface o f which would be open to i n f e c t i o n . I t was observed that when l e v e l s o f v i r u s w i t h i n the kidney i n c r e a s e d s u b s t a n t i a l l y on the f o u r t h to f i f t h day, a sudden r i s e i n v i r u s l e v e l s w i t h i n bladder t i s s u e was observed between four and seven days. During the course o f examination o f i n f e c t e d t i s s u e s using a v a r i e t y of h i s t o l o g i c a l techniques, i t was d i f f i c u l t to recognize any overt patholo-g i c a l changes i n s e c t i o n s o f the various t i s s u e s examined with the exception of the br a i n using the H and E method o f s t a i n i n g . A f t e r the i n f e c t i o n o f mice with aerosols c o n t a i n i n g Powassan v i r u s , t i t r a t i o n o f Powassan v i r u s l e v e l s i n various t i s s u e s plus the v i s u a l i z a t i o n of Powassan v i r u s s p e c i f i c f o c i f i r s t i n c r o s s - s e c t i o n s o f mouse lung would seem to i m p l i c a t e the mouse lung as the primary s i t e o f v i r a l i n v a s i o n and m u l t i p l i c a t i o n . In concert with the r i s e o f Powassan v i r u s i n the blood stream, fl u o r e s c e n t - a n t i b o d y s t a i n e d s a g i t t a l s e c t i o n s o f mouse b r a i n three days a f t e r aerosol exposure showed Powassan v i r u s s p e c i f i c fluorescence i n cuboidal e p i -68 thelium o f the choroid plexus. There are four places where the wall o f the bra i n r e t a i n s i t s embryonic c h a r a c t e r as a t h i n non-nervous e p i t h e l i u m . This p a r t o f the b r a i n wall i s the lamina e p i t h e l i a l i s . The pia mater which covers i t i s extremely v a s c u l a r and otherwise modified to form a choroid plexus. The lamina e p i t h e -l i a l i s i s c l o s e l y j o i n e d to the choroid plexus and the whole i s c a l l e d the t e l a choroidea, or l e s s e x a c t l y , choroid plexus. These choroid plexuses are found i n the roof o f the t h i r d and fourth v e n t r i c l e s and i n a pa r t o f the wall o f the two l a t e r a l v e n t r i c l e s . In each case the t e l a choroidea i s much f o l d e d and invaginated i n t o the ven-t r i c l e so that the fr e e surface exposed to the v e n t r i c u l a r f l u i d i s large with tortuous vessels and a r i c h c a p i l l a r y net. In animals repeatedly i n j e c t e d i n t r a v e n o u s l y with v i t a l dyes, such as trypan blue, the e p i t h e l i u m o f the choroid plexus stores large amounts o f the dye. Also i n the p e r i v a s c u l a r connective t i s s u e core o f the plexus are many f i x e d macrophages, which store l a r g e amounts of the dye (Bloom and Fawcett, 1968). On the boundary between adjacent e p i t h e l i a l c e l l s i n e l e c t r o n micrographs, there i s a juxtaluminal f u n c t i o n a l complex that appears to seal the i n t e r c e l l u l a r space. The c a p i l l a r i e s beneath the e p i t h e l i u m are u n l i k e those elsewhere i n the b r a i n , i n that they are t h i n walled and have f e n e s t r a -t i o n s o r pores c l o s e d by t h i n diaphragms. The j u n c t i o n s between e n d o t h e l i a l c e l l s a l s o appear to be more permeable. A p r o t e i n e x t r a c t e d from horse r a d i s h (peroxidase) when i n j e c t e d i n t o the femoral vein o f mice was demonstrable wi t h i n the choroid plexus. Peroxidase was pinocytosed by the f e n e s t r a t e d endothelium but apparently d i d not penetrate the f e n e s t r a l diaphragms. I t i s as y e t uncertain whether endo-t h e l i a l c e l l j u n c t i o n s ( A l b r e c h t , 1968) completely excluded t h i s p r o t e i n , but 69 enough crossed the endothelium, presumably by v e s i c u l a r t r a n s p o r t to pene-t r a t e the p e r i v a s c u l a r basement membrane. Peroxidase then moved between e p i t h e l i a l c e l l s u n t i l stopped by c o n i c a l s t r u c t u r e s o f the i n t e r s p a c e near the v e n t r i c u l a r s u r f a c e (Brightman, 1967). Smaller p a r t i c l e s , such as f e r r i t i n , can be t r a c e d s h o r t l y a f t e r intravenous i n j e c t i o n s , as they make t h e i r way through the system o f endothe-l i a l v e s i c l e s and through the basement membrane. A f t e r i n j e c t i n g l a r g e doses o f carbon i n t o rats and mice, i t was found i n the f e n e s t r a t e d endothelium i n many organs. However, the concentration of carbon was never hig h , presumably because o f the competition o f the r e t i c u l o e n d o t h e l i a l system i n c l e a r i n g carbon p a r t i c l e s . In l a t e r experiments p e r f u s i o n of an organ with c o l l o i d s o l u t i o n s i n order to minimize the e f f e c t s caused by the r e t i c u l o e n d o t h e l i a l system, uptake of p a r t i c u l a t e matter by the endothelium could be much more e a s i l y demonstrated. Impounding o f large d r o p l e t s even measuring several microns by the e n d o t h e l i a l c e l l s of large vessels and c a p i l l a r i e s o f the b r a i n was r e g u l a r l y observed. This might e x p l a i n how l a r g e molecules the s i z e of v i r u s e s and r i c k e t t s i a are taken up by e n d o t h e l i a l c e l l s . Phago-c y t o s i s i n a broad sense i s a common phenomenon in e n d o t h e l i a l c e l l s . How-ever, the phenomenon i s d i f f e r e n t from that performed f o r example by Kupffer c e l l s , because i t leads p r i m a r i l y to t r a n s p o r t across the c e l l r a t h e r than to i n t r a c e l 1 u l a r storage and d i g e s t i o n ( A l b r e c h t , 1968). Fluorescence was not detected i n the v a s c u l a r endothelium during these s t u d i e s . However, i f the v i r u s was merely passed through these c e l l s i t i s not l i k e l y that they would show s p e c i f i c f l u o r e s c e n c e . Other arbo-vi r u s e s which cause c e n t r a l nervous system disease where i n f e c t i o n o f the va s c u l a r endothelium could not be demonstrated are Japanese B e n c e p h a l i t i s v i r u s , t i c k - b o r n e e n c e p h a l i t i s v i r u s , Semliki Forest v i r u s and dengue v i r u s ( A l b r e c h t , 1968). 70 N i r e_t al_ (1965) exposed mice to aerosols o f West N i l e v i r u s (also a member o f the group B a r b o v i r u s e s ) . T h e i r r e s u l t s i n one experiment show that West N i l e v i r u s i n f e c t i v i t y appeared i n the lung at day zero and was at i t s height a t 72 hours at 4.5 l o g . I t appeared f i r s t i n the blood at 48 hours at 0.2 l o g , i n the b r a i n at 48 hours at 0.1 l o g and i n the nasal e p i -thelium at 72 hours at 0.1 l o g . These f i n d i n g s are s i m i l a r to the Powassan v i r u s s t u d i e s contained i n t h i s t h e s i s . That i s , primary s i t e o f m u l t i p l i c a t i o n i n the lung r e s u l t i n g i n a viremia i n 24 hours followed by the presence o f the v i r u s i n other t i s s u e s a t 48 hours. In a second experiment, N i r ejt al_ d e scribed a s i t u a t i o n where 2.6 log o f West N i l e v i r u s appeared i n the o l f a c t o r y bulb o f the b r a i n at 36 hours a f t e r i n o c u l a t i o n and a t t a i n e d a l e v e l o f 4.5 l o g 48 hours a f t e r i n o c u l a t i o n and i n the cerebellum at l e s s than 1 l o g 48 hours a f t e r i n o c u l a t i o n . Unfor-t u n a t e l y no record o f viremia or the appearance o f v i r u s i n other t i s s u e s was inc l u d e d . Judging from past experience, the l e v e l s o f v i r u s and the time of appearance as presented i n the second experiment would i n d i c a t e that the mice had re c e i v e d a s u b s t a n t i a l l y higher dose of v i r u s than they d i d i n the f i r s t experiment. This would have the e f f e c t o f advancing the sequence o f events and a m p l i f y i n g the l e v e l s of v i r u s t i t r a t e d i n each t i s s u e (Danes e t a l , 1962). It i s a l s o i n t e r e s t i n g that i n the f i r s t experiment West N i l e v i r u s was present i n the b r a i n 24 hours before i t was observed i n the nasal e p i -thelium. Also that under the c o n d i t i o n s of the second experiment, 4- to 6 log was present i n the b r a i n at 48 hours, whereas i n the f i r s t experiment only 0.1 l o g was present i n the b r a i n at 48 hours. N i r e_t al_ concluded that West N i l e v i r u s invades the c e n t r a l nervous system o f the mouse through i n f e c -t i o n of the nerves o f the o l f a c t o r y mucosa. 71 The pathogenesis o f Powassan v i r u s i n mice a f t e r airborne i n f e c t i o n occurred i n the f o l l o w i n g sequence: i n f e c t i o u s v i r u s p a r t i c l e s were a s p i r a t e d i n t o the lung, invaded and m u l t i p l i e d w i t h i n the t i s s u e s o f t h i s organ, estab-l i s h i n g a viremia 24 hours a f t e r i n o c u l a t i o n . When the l e v e l o f Powassan vi r u s w i t h i n the blood a t t a i n e d a thr e s h o l d t i t r e o f approximately 3 logs (Japanese B e n c e p h a l i t i s 1.8 l o g per 0.03 ml. i n mice, Huang and Wong, 1963), i n f e c t i v i t y appeared i n other t i s s u e s i n c l u d i n g the b r a i n . Fluorescent antibody stu d i e s i n d i c a t e that the e p i t h e l i a l c e l l s o f the ch o r o i d plexus may be i m p l i c a t e d i n the spread o f the viru s to the cent r a l nervous system. Although fluorescence i n vascular endothelium was not ob-served, (". . . the l e v e l o f m u l t i p l i c a t i o n of v i r u s w i t h i n v a s c u l a r endo-thelium may be so low as to escape d e t e c t i o n by the n o t - v e r y - s e n s i t i v e f l u o r e s -cent-antibody technique ..." A l b r e c h t , 1968), passage o f v i r u s by way o f the vas c u l a r endothelium may als o be o f importance i n the i n f e c t i o n o f the ce n t r a l nervous system. Studies would i n d i c a t e that the vi r u s i s capable o f m u l t i p l y i n g w i t h i n a v a r i e t y o f t i s s u e s , notably t i s s u e s which have a high e p i t h e l i a l c e l l content. Powassan v i r u s m u l t i p l i e s w i t h i n the b r a i n o f the mouse to extremely high l e v e l s e v e n t u a l l y causing death o f the mouse. Fluorescent-antibody s t u d i e s i n d i c a t e wide involvement o f t i s s u e s o f the lung and b r a i n , with l e s s s p e c i f i c f l u o r e s c e n c e being seen i n other organs o f the mouse. E l e c t r o n micrographs reveal the presence o f v i r u s s p e c i f i c s t r u c -tures i n macrophages o f the lung and i n the nasal e p i t h e l i a l c e l l s o f mice i n f e c t e d by i n t r a n a s a l i n s t i l l a t i o n o f v i r u s . (Nelson, 1969a) These s t r u c t u r e s are reminiscent o f s i m i l a r s t r u c t u r e s observed by Grimley e t al_ (1968), Tan (1970), who studied the morphogenesis of Semliki Forest v i r u s and Venezuelan equine encephalomyelitis v i r u s (Garcia-Tamayo, 1971), both r e p r e s e n t a t i v e s o f the group A arboviruses. 72 TABLE 1 ANTIGENIC GROUPS OF ARBOVIRUSES** GROUP ABBREVIATION A A B B C* C A f r i c a n horse sickness AHS Anopheles A ANA Bakau BAK Bluetongue BLU Bunyamwera* BUN Bwamba* BWA C a l i f o r n i a * CAL Capim* CAP Changuinola CGL E p i z o o t i c hemorrhagic disease o f deer EHD Guama* GMA Koongol* K00 P a t o i s * PAT Phlebotomus ( s a n d f l y ) fever PHL P i r y PIRY Quaranfil QRF Simbu* SIM Tacaribe TCR Timbo TIM Turlock TUR V e s i c u l a r s t o m a t i t i s VSV * The Bunyamwera supergroup inc l u d e s members of the f o l l o w i n g nine groups, among which some i n t e r r e l a t i o n s have been shown: Bunyamwera, Bwamba, Group C, C a l i f o r n i a , Capim, Guama, Koongol, Patois and Simbu. ** ACAV, 1969 73 TABLE 2 ANTIGENIC CLASSIFICATION OF ARBOVIRUSES** GROUP GROUP A A f r i c a n h o r s e s i c k n e s s Anopheles A Anopheles B B Bakau Bluetongue Bunyamwera Supergroup Bunyamwera Bwamba C Cal i f o r n i a Capim Guama Koongol Pato i s Simbu Tete Unassigned Changui no!a Congo E p i z o o t i c hemorrhagic d i sease Ganjam Hughes Ka isod i Kemerovo Mapputta Mossuri1 Nyando Phlebotomus f e v e r Qalyub Quaranf i 1 Timbo Tur lock Uukuniemi V e s i c u l a r s t o m a t i t i s Ungrouped Tacar ibe* * Evidence f o r an ar th ropod c y c l e i n nature i s scant f o r a l l the v i r u s e s i n t h i s groupo ** ACAV, 1970 Matar iya*** Palyam*** *** ACAV, 1971 TABLE 3 PROTOTYPE STRAINS OF THE RUSSIAN SPRING-SUMMER COMPLEX OF TICK-BORNE GROUP B ARBOVIRUSES Prototype Virus I n i t i a l I s o l a t i o n Name S t r a i n Year Location Geographical D i s t r i b u t i o n Tick Vector Mammal Reservoir Syndrome i n Man Louping i l l (LI) O r i g i n a l 1929 Scotland Scotland, U l s t e r I. r i c i n u s v o l e s , mice A s e p t i c meningitis sheep Russian spring-summer S o f j i n e n c e p h a l i t i s (RSSE) Omsk hemorrhagic f e v e r (OMSK) Negishi (NEG) Tick-borne enceph-a l i t i s (TBE) Langat (LGT) Kyasanur Forest Disease (KFD) Powassan (POW) Kubrin 1937 Far eas-tern U.S.S.R. 1947 Omsk Oblast U.S.S.R. (Eu- I. p e r s u l -ropean S i b e r - catus i a ) Far East O r i g i n a l 1948 Japan Absett-arov Hypr 1951 Lenin-grad Oblast U.S.S.R. ( S i b e r i a ) Japan U.S.S.R. (European) A u s t r i a , 1953 Czecho-Hansalova 1948 Sl o v a k i a Czechoslova-Kumlinge 1959 Finland k i a , Finland Hungary, Po-land, Sweden TP21 W371 1956 Malaysia Malaysia LB 1957 Mysore, India 1958 Ontario, Canada India Canada and U.S.A. D. p i c t u s I. r i c i n u s & I. p e r s u l -catus I. r i c i n u s f o r e s t rodents f o r e s t rodents f o r e s t rodents goats (milk borne) f o r e s t rodents I. granulatus I. granulatus H. s p i n i g e r a and o t h e r Hae-maphysalis sp. i . cookei f o r e s t rodents monkeys f o r e s t rodents f o r e s t rodents E n c e p h a l i t i s Fever, hemorrhages E n c e p h a l i t i s A s e p t i c meningitis Meningoenceph-al i t i s Hemorrhagic f e v e r Encephali t i s 75 TABLE 4 MINIMUM LETHAL DOSES FOR VARIOUS ROUTES OF INFECTION ROUTE OF INFECTION DOSAGE MICLD 5 Q* I n t r a c e r e b r a l i n o c u l a t i o n Intravenous i n o c u l a t i o n Subcutaneous i n o c u l a t i o n I n t r a p e r i t o n e a l i n o c u l a t i o n Aerosol i n o c u l a t i o n Intranasal i n s t i l l a t i o n Per Os i n s t i l l a t i o n 1 1 18 98 640 10,000 No i n f e c t i o n a f t e r 3 x 10' * MICLDgg = mouse i n t r a c e r e b r a l LD^g = the minimum amount of Powassan v i r u s per ml. requ i r e d to cause death i n 50 per cent o f three- to four-week-old mi ce. TABLE 5 CROSS-INFECTION RECORD Days Deaths Due To Post-Exposure I n f e c t i o u s Aerosol Contact 8 9 9 9 10 11 11 1 16 1 NS 17 18 2 19 4 20 4 21 22 1 23 2 24 1 NS Aerosol Deaths 30/30 Contact Deaths 13/30 NS - n o n - s p e c i f i c -- may or may not have been i n f e c t e d ; 43 per cent o f co n t r o l mice i n f e c t e d because o f intimate contact with a e r o s o l -i n f e c t e d mice. TABLE 6 LEVELS OF POWASSAN VIRUS IN VARIOUS MOUSE TISSUES AFTER INTRANASAL INSTILLATION OF 10 MINLD c nOF VIRUS D a y s A f t e r I n f e c t i o n Mouse Tissue 0 1 2 3 4 5 6 7 Turbinates 0 2.76 2. 76 3.76 5.28 5.28 5.28 5.0 Lung 3.0 4.0 5. .28 6.28 6.28 5.28 5.28 4.69 Blood 0 0 3. 72 4.98 4.77 3.48 2.97 2.97 Spleen 0 0 0 5.69 4.69 4.28 3.52 3.28 Brain 0 0 3. ,3 4.52 6.0 8.0 8.77 9.28 Figures presented represent the average of three separate experiments f o r each t i s s u e examined. TABLE 7 LEVELS OF POWASSAN VIRUS  IN 10 PER CENT TISSUE HOMOGENATES LOG ,„/ML. D a y A f t e r A e r o s o 1 I n o c u 1 a t i o n Tissue 0 1 2 3 4 5 6 7 8 Turbinates 0 + 0 2.0 + 4.5 5.8 5.0 + Lung 0 1.8 2.8 4.5 4.8 5.3 4.8 5.2 3.2 Thymus 0 0 + 2.8 2.8 3.3 3.8 4.2 4.8 Blood 0 0 3.2 4.1 4.0 + 4.0 4.1 2.9 Spleen 0 0 1.8 2.8 4.3 4.5 4.2 2.8 4.2 Li ver 0 0 0 + 1.8 2.0 2.3 2.0 0 Ki dney 0 0 + 2.8 2.3 5.0 4.0 4.0 3.0 Gut 0 0 0 0 0 + 0 0 0 Sk e l e t a l Muscle 0 0 0 2.0 + 3.3 3.0 4.0 2.8 Cardiac Muscle 0 0 0 + 3.0 3.0 2.8 4.2 3.0 Smooth Muscle 0 0 0 0 0 3.0 4.3 6.0 3.0 Brain 0 0 + 4.0 4.0 5.0 7.8 8.5 7.8 ures presented represent the average of f i v e separate experiments f o r each t i s s u e examined, virus present, but too low to quan t i t a t e TABLE 8 BIOLOGICAL DECAY OF POWASSAN VIRUS SUSPENSIONS IN THE DARK AND UNDER ULTRAVIOLET IRRADIATION Suspension Condition T i m e I n M i n u t e s 0 30 60 90 120 150 180 Dark 21°C L 7.77 H 8.0 7.8 ± 0.24* 0 UV 20 micro-watts/cm 21 6C 7.77 8.0 7.9 ± 0.24* 7.28 7.77 7.5 ± 0.41 15 7.52 7.77 7.6 ± 0.24 30 7.28 7.52 7.4 ± 0.24 45 6.77 7.0 6.9 ±0.24 60 6.55 6.77 6.7 ± 0.24 75 6.0 6.52 6.3 ± 0.24 5.52 6.0 5.77 ± 0.24 2.52 3.77 3.69 ± 0.24 2.77 3.27 3.0 ± 0.29 + + + 6.52 7.0 6.8 ± 0.34 90 0 0 0 * = Mean l o g 1 Q LD 5 0/ml. based on three measurements with ± 2 sigma l i m i t s . + = Virus present but too low to qu a n t i t a t e . L = Lowest value. H = Highest value. TABLE 9 BIOLOGICAL DECAY OF POWASSAN VIRUS AEROSOLS AT LOW, INTERMEDIATE  AND HIGH RELATIVE HUMIDITIES AND CONSTANT TEMPERATURE LOG 1 Q LD 5 Q/ML. OF IMPINGER FLUID Aerosol T i m e I n H o u r s Condition 0 1 2 3 4 5 20% RH L 5.27 4.0 3.77 4.0 3.77 4.0 21°C H 5.76 4.27 4.27 4.27 4.0 4.0 5.02 + 0. 41* 4.1 - 0.24 4.1 t 0.24 4.1 - 0.24 3.94 - 0.24 4.0 - 0.0 50% RH 4.77 3.0 3.0 3.0 3.0 2.77 21°C 5.0 3.52 3.27 3.27 3.27 3.27 4.85 + 0. 24 3.35 - 0.47 3.1 t 0.24 3.2 - 0.24 3.2 - 0.24 3.02 - 0.41 80% RH 4.52 3.52 3.52 3.52 3.52 3.27 21°C 5.0 4.0 3.77 3.77 3.77 3.52 4.85 + 0. 24 3.85 - 0.47 3.6 - 0.24 3.68 - 0.24 3.6 - 0.24 3,44 - 0.24 Tracer 4.98 4.93 4.9 4.95 4.89 4,89 21°C 5.18 5.0 5.0 5.08 4.95 4.98 5.06 + 0. 7 4.97 - 0.69 4.94 - 0.23 5.0 - 0.41 4.92 - 0.30 4.92 - 0.18 * = Mean l o g 1 Q LDj-g/ ml. based on three measurements and - 2 Sigma l i m i t s . L = Lowest value, H = Highest value 00 o TABLE 10 BIOLOGICAL DECAY OF POWASSAN VIRUS AEROSOLS IN THE DARK AND UNDER ULTRAVIOLET IRRADIATION Aerosol T i m e I n M i n u t e s Condition 0 60 120 180 Dark 20% RH 21°C L 4.0 3.27 3.0 3.27 H 5.0 4.0 4.0 4.0 4.69 - 0.95* 3.78 - 0.84 3.76 - 0.88 3.79 - 0.93 T i m e I n M i n u t e s 0 5 10 , 15 30_ U.V. 400 microwatts/cm 2 4.77 + 0 0 0 20% RH 21°C 5.0 + 0 0 0 4.9 - 0.63* 1.79 0 0 0 Mean l o g 1 Q LD 5 0/ml. based on three measurements and - 2 Sigma l i m i t s . Virus present but too low to q u a n t i t a t e . Lowest value. Highest value 82 FIGURE 1 Diagram o f aerosol s u i t e . 83 84 FIGURE 2 Diagram o f the aerosol animal exposure u n i t and Legend the aerosol aging drum. A 500 l i t e r aerosol drum L a l l glass impinger B aerosol mixing tube M animal exposure conta i n e r C water manometer N dry bulb thermometer D ai r flow meter P wet bulb thermometer E a i r flow r e g u l a t o r R valves F p a r t i c u l a t e f i l t e r S heat exchanger thermometer 6 heat exchanger T heat exchanger drain H h o t / c o l d water mixer r e g u l a t o r U hot water l i n e I aerosol humidity c o n t r o l tank V c o l d water l i n e J C o l l i s o n atomizer .=o= 4 B OL 8R I t 1 BR •OL BR N AEROSOL APPARATUS DIAGRAM SEE LEGEND oo FIGURE 3 Growth curve o f Powassan v i r u s i n s u c k l i n g mouse b r a i n . 87 88 FIGURE 4 Levels o f Powassan v i r u s i n various mouse t i s s u e s a f t e r i n t r a n a s a l i n s t i l l a -t i o n o f 10 MINLD c n o f Powassan v i r u s . 68 90 FIGURE 5 Curve of the r e t e n t i o n of Powassan v i r u s i n the lungs o f mice a f t e r aerosol i n o c u l a t i o n . 0 5 10 15 TIME IN MINUTES 92 FIGURE 6 B i o l o g i c a l decay of Powassan v i r u s suspensions i n the dark and under u l t r a -v i o l e t i r r a d i a t i o n . TIME IN HOURS 94 FIGURE 7 B i o l o g i c a l decay of Powassan v i r u s aerosols at low, intermediate and high r e l a t i v e humidites and at a constant temperature o f 21°C. T r a c e r = B a c i l l u s s u b t i l ! u s (var. n i g e r ) spores which are not s u s c e p t i b l e to b i o l o g i c a l decay. 2 -r1 0 "I 1 1 I 2 3 TIME IN HOURS T 4 T 5 96 FIGURE 8 B i o l o g i c a l decay o f Powassan v i r u s aerosols at various r e l a t i v e h u m i d i t i e s . 97 98 FIGURE 9 B i o l o g i c a l decay of Powassan v i r u s aerosols i n the dark and under u l t r a v i o l e t i r r a d i a t i o n . 99 TIME IN HOURS 100 FIGURE 10 Levels of Powassan v i r u s i n various t i s s u e s a f t e r aerosol i n o c u l a t i o n vi rus. L 0 0 N HOUSE INTRACEREBRAL LOgQ/ ML. L 0 G K > INTRACEREBRAL L O w / W . . n r •] L O a m MOUSE I N T R A C E R E B R A L L O J Q / M L . t O O 0 MOUSE INTRACEREBRAL L D Q Q / M L . LOa w MOUSE INTRACEREBRAL LDgg / « W MOUSE I N T R A C E R E B R A L L O Q Q / — N w * ta 102 PLATE 1 Photograph o f the aerosol aging apparatus w i t h i n the climate control room o f the aerosol s u i t e . 103 104 PLATE 2 Photograph o f animal exposure apparatus w i t h i n the climate room o f the aerosol s u i t e . 105 106 PLATE 3 Photograph o f researcher wearing p r o t e c t i v e c l o t h i n g plus the p o s i t i v e pressure s a f e t y hood which i s attached to the separate, f i l t e r e d , con-d i t i o n e d a i r supply w i t h i n the climate room o f the aerosol s u i t e . 107 108 PLATE 4 Photograph of the modified H o r s f a l l cages used f o r the containment o f mice exposed to i n f e c t i o u s aerosols o f Powassan v i r u s . The a i r e n t e r i n g and l e a v i n g the u n i t i s f i l t e r - s t e r i l i z e d . The a i r l e a v i n g the uni t s i s a l s o subjected to u l t r a v i o l e t i r r a d i a t i o n o f 1600 microwatts/cm plus a i r i n c i n -e r a t i o n a t 450°F. 110 PLATE 5 Photograph of the s p e c i a l needle used f o r the i n s t i l l a t i o n of Powassan vi r u s i n t o the g a s t r o i n t e s t i n a l t r a c t o f mice. 112 PLATE 6 Cross-section of un i n f e c t e d mouse lung, s t a i n e d with H and E, magnifica-t i o n 320 x. PLATE 7 Cros s - s e c t i o n o f Powassan v i r u s i n f e c t e d mouse lung f i v e days a f t e r aerosol i n o c u l a t i o n , s t a i n e d with H and E, ma g n i f i c a t i o n 320 x. 113 114 PLATE 8 Cross - s e c t i o n o f Powassan v i r u s i n f e c t e d mouse lung f i v e days a f t e r aerosol i n o c u l a t i o n , showing t h i c k e n i n g o f a l v e o l i e p i t h e l i u m and accumulation o f macrophages, s t a i n e d with H and E, ma g n i f i c a t i o n 640 x. PLATE 9 S a g i t t a l s e c t i o n o f uninfected mouse cerebellum, s t a i n e d with H and E, magni-f i c a t i o n 640 x. 116 PLATE 10 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d mouse cerebellum three days a f t e r aerosol i n o c u l a t i o n , showing the beginning o f c e l l u l a r degeneration; s t a i n e d with H and E, m a g n i f i c a t i o n 320 x. PLATE 11 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d mouse cerebellum e i g h t days a f t e r aerosol i n o c u l a t i o n . Note widespread c e l l u l a r degeneration; s t a i n e d with H and E, m a g n i f i c a t i o n 320 x. 118 PLATE 12 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d mouse cerebrum s i x days a f t e r aerosol i n o c u l a t i o n showing c e l l u l a r degeneration and i n f i l t r a t i o n o f inflammatory c e l l s ; s t a i n e d with H and E, m a g n i f i c a t i o n 320 x. PLATE 13 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d mouse b r a i n at the s i t e o f cleavage planes between adjacent p o r t i o n s o f the b r a i n . Note c e l l u l a r degeneration plus inflammatory c e l l i n f i l t r a t i o n ; s t a i n e d with H and E, ma g n i f i c a t i o n 320 x. PLATE 14 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d mouse b r a i n at the s i t e o f cleavage planes between adjacent p o r t i o n s o f the b r a i n . Note c e l l u l a r degeneration plus inflammatory c e l l i n f i l t r a t i o n ; s t a i n e d with H and E, ma g n i f i c a t i o n 320 x. 119 120 PLATE 15 Cros s - s e c t i o n o f Powassan v i r u s i n f e c t e d mouse lung two to three days a f t e r aerosol i n o c u l a t i o n , showing Powassan v i r u s s p e c i f i c fluorescence i n lung e p i t h e l i a l c e l l s . Stained with FITC conjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 640 x. PLATE 16 Cros s - s e c t i o n o f Powassan v i r u s i n f e c t e d mouse lung f i v e days a f t e r aerosol i n o c u l a t i o n . Note widespread Powassan v i r u s s p e c i f i c f l u o r e s c e n c e . Stained with FITC conjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , magnifi-c a t i o n 640 x. 121 122 PLATE 17 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d mouse b r a i n showing c e l l s o f the choroid plexus of the mouse as they protrude i n t o the fourth v e n t r i c l e o f the brain c l o s e to the cerebellum. Stained with FITC conjugated Powas-san v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 160 x. PLATE 18 S a g i t t a l s e c t i o n of Powassan v i r u s i n f e c t e d c e l l s o f the choroid plexus o f the mouse three days a f t e r aerosol i n o c u l a t i o n . Note Powassan v i r u s s p e c i f i c fluorescence o f cuboidal c e l l s o f the choroid plexus. Stained with FITC con-jugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 640 x. 123 124 PLATE 19 S a g i t t a l s e c t i o n o f Powassan v i r u s i n f e c t e d c e l l s o f the choroid plexus o f the mouse three days a f t e r aerosol i n o c u l a t i o n , showing Powassan v i r u s s p e c i f i c f l u o r e s c e n c e . Stained with FITC conjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 640 x. PLATE 20 S a g i t t a l s e c t i o n o f Powassan vir u s i n f e c t e d c e l l s o f the choroid plexus o f the mouse three days a f t e r aerosol i n o c u l a t i o n , showing Powassan v i r u s s p e c i f i c f l u o r e s c e n c e . Stained with FITC conjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 640 x. 126 PLATE 21 Sag g i t a l s e c t i o n o f Powassan vir u s i n f e c t e d mouse b r a i n at .the i n t e r f a c e s between the archnoid and pi a mater; the pi a mater and cerebral cortex three days a f t e r aerosol i n o c u l a t i o n . Note points o f fluorescence i n c e l l s of the cerebral cortex. Stained with FITC conjugated Powassan v i r u s s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 640 x. PLATE 22 S a g i t t a l s e c t i o n o f Powassan vir u s i n f e c t e d mouse b r a i n s i x days a f t e r aerosol i n o c u l a t i o n showing widespread Powassan v i r u s s p e c i f i c f luorescence i n t i s s u e s o f the cerebrum. Stained with FITC conjugated Powassan s p e c i f i c r a b b i t gamma g l o b u l i n , m a g n i f i c a t i o n 320 x. 127 128 PLATE 23 E l e c t r o n micrograph o f what i s b e l i e v e d to be a macrophage o f the lung three days a f t e r i n t r a n a s a l i n s t i l l a t i o n o f Powassan v i r u s , showing what appear to be phagolysosomes s t r u c t u r e s (arrows) which o r d i n a r i l y destroy f o r e i g n material a s p i r a t e d i n t o the lung. Stained with osmium t e t r o x i d e , m a g n i f i c a t i o n approximately 50,000 x. 129 130 PLATE 24 E l e c t r o n micrograph o f nasal e p i t h e l i u m s i x days a f t e r i n t r a n a s a l i n s t i l l a -t i o n o f Powassan v i r u s . Note s t r u c t u r e s which appear to be v i r u s p a r t i c l e s (arrows) arranged around vacuoles w i t h i n the cytoplasm of e p i t h e l i a l c e l l s . S tained with osmium t e t r o x i d e , m a g n i f i c a t i o n approximately 50,000 x. 132 PLATE 25 E l e c t r o n micrograph o f nasal e p i t h e l i u m s i x days a f t e r i n t r a n a s a l i n s t i l l a -t i o n o f Powassan v i r u s . Note s t r u c t u r e s which appear to be v i r u s p a r t i c l e s arranged around vacuoles w i t h i n the cytoplasm o f e p i t h e l i a l c e l l s . Stained with osmium t e t r o x i d e , m a g n i f i c a t i o n approximately 50,000 x. 134 PLATE 26 E l e c t r o n micrograph of nasal e p i t h e l i u m s i x days a f t e r i n t r a n a s a l i n s t i l l a -t i o n o f Powassan v i r u s . Note s t r u c t u r e s which appear to be v i r u s p a r t i c l e s arranged around vacuoles w i t h i n the cytoplasm o f e p i t h e l i a l c e l l s . 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