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Bunyamwera virus replication in arthropod and vertebrate tissue. Peers, Robert Ross 1971

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BUNYAMWERA VIRUS REPLICATION IN ARTHROPOD AND VERTEBRATE TISSUE by ROBERT ROSS PEERS B.Sc. University of B r i t i s h Columbia 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1971 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 r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g 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 g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Co lumbia V a n c o u v e r 8, Canada Department o f ^""] i Q y7\ V? f 0\ 0 Abstract Bunyamwera (BUN) virus m u l t i p l i e d r e a d i l y i n mosquitoes following intrathoracic i n j e c t i o n and also after imbibing'-an i n f e c t i v e blood meal. This agent also m u l t i p l i e d following inoculation of human and avian c e l l s i n tissue cultures, with production of cytopathic effects. Both i n arthropod and vertebrate tissues, enveloped vi r i o n s 8^ nm diameter were v i s u a l i z e d by electron microscopic observation of tissues collected after maximum v i r a l p r o l i f e r a t i o n was attained. 2 2 Following intrathroacic i n j e c t i o n of 10 " mouse LD„„ of BUN 50 virus into groups of wild-caught mosquitoes comprising both Aedes canadensis and A. vexans, increments of i n f e c t i v i t y were f i r s t detected 5.2 i n s a l i v a r y glands and gut at 3 days, and maximum t i t r e s of 10 * mouse ID,., per organ were attained i n salivary glands at 10 days. However, 50 the virus content of legs, which provided a convenient means of sampling hemocelic f l u i d , increased at 2 days. Virus transmission by b i t i n g mice was demonstrated with mosquitoes injected 10 days previously, but not after shorter intervals. No virus r e p l i c a t i o n was demonstrated follow-h 0 ing ingestion of 10 " mouse of BUN virus i n a blood meal. Aedes aegypti mosquitoes re a d i l y supported the r e p l i c a t i o n 3 3 of BUN virus following i n j e c t i o n with 10 mouse ID,__ or imbibing 50 k.6 of 1 0 * mouse L D ^ Q - After i n j e c t i o n , virus t i t r e s of whole mosquitoes 1 7 declined to 1 0 mouse L D ^ Q A ^ 1 2 hours, followed by an increase to a 5 0 peak amount of 10 mouse LD,_„ at 2 days. After feeding, virus was 50 f i r s t detected i n legs and sa l i v a r y glands at k days, and attained 5 0 maximum t i t r e s of 10 mouse L D C _ i n sal i v a r y glands at 10 days. 50 Transmission of virus to mice was effected by A. aegypti following feeding and i n j e c t i o n 10 days previously, but not at e a r l i e r i n t e r v a l s . Following exposure of whole gut cultures of adult A. aegypti mosquitoes to 10^" ^ mouse L D maximum yields of 10^"^ mouse L D per p(J 50 ml. were observed after k days incubation at 29 ° C , after an i n i t i a l 1 8 decline of i n f e c t i v i t y to 10 * mouse L D . _ _ . at 12 hours. 50 Enveloped v i r i o n s with cores k*? nm diameter and t o t a l d i a -meters 80 to 100 nm were observed within vacuoles and l i n i n g vacuolar membranes of sal i v a r y glands and gut c e l l s of A. aegypti mosquitoes 10 days or more after i n f e c t i o n with BUN virus. Wo p a r t i c l e s were observed e a r l i e r , despite high virus t i t r e s k days or more after i n j e c -t i o n . After inoculation of continuous l i v e tissue cultures of human 6 5 epidermoid corcinoma c e l l s (H.Ep. 2) with 10 mouse L D ^ Q? the highest 7 0 amount of virus produced was 10 * mouse L D ^ Q per ml. c e l l suspension after 2k hours incubation at 37 ° C . Maximum yields of BUN virus (10^* 2 mouse L D , - Q per ml. c e l l suspension) were attained 2k hours after inocu-5 2 l a t i o n of primary chick embryo f i b r o b l a s t monolayers with 10 mouse On 1 4 . - 4 . <, n r . 5 . 0 1 ( 1 1 1 / m i 1 n u i r i f * i i [ > ; - H i . i f i r i x 1 . - s / rtT ^ "4~ 50 L D C N follow ng incubation at 37 C . However, a peak t i t r e of 10 3 7 mouse L D . was attained 3 days after inoculation with 10 mouse L D ^ ^ 50 50 i n cultures incubated at 29 ° C . Before an increment of virus t i t r e was observed i n f e c t i v i t y declined to zero during the i n i t i a l k hours after inoculation of cultures incubated at 37 ° C , and a tenfold decline of i n f e c t i v i t y was noted i n cultures incubated at 29 ° C . Enveloped v i r i o n s with t o t a l diameter 8h nm which contained electron-dense nucleoids hh nm diameter were observed e x t r a c e l l u l a r l y i n t h i n sections of chick embryo fib r o b l a s t s infected 12 hours pre-viously with BUN vi r u s . These p a r t i c l e s were released by budding. Precursor p a r t i c l e s hi nm diameter were associated with i n t r a c e l l u l a r membranes i n occasional c e l l s sectioned at h hours. E x t r a c e l l u l a r v i r i o n s released one day after inoculation of H.Ep. 2 cultures were tagged by f e r r i t i n - l a b e l l e d anti-BUN antibody. Enveloped vi r i o n s with mean diameters 100 nm were observed i n suspensions of suckling mouse brain infected with BUM virus and stained negatively with phosphotungstic acid. These results show c l e a r l y that BUN virus exhibits the essential b i o l o g i c a l and morphological characteristics of a mosquito -borne arbovirus. i v . Table of Contents Page INTRODUCTION AND LITERATURE REVIEW Arboviruses a. General characteristics 1 b. C l a s s i f i c a t i o n 1 c. Infection cycles . 2 d. Replication i n mosquitoes 3 e. Electron microscopy of r e p l i c a t i o n 5 General Characteristics of Bunyamwera and the Bunyamwera Group a. C l a s s i f i c a t i o n 9 b. History . 9 c. Replication of Bunyamwera l i MATERIALS AND METHODS Virus ik Virus Assay lh Serology 15 Preparation of Anti-Bunyamwera Serum 15 Viremia Experiments a. Rabbits l 6 b. Mice l 6 Tissue Culture 17 Infection of CEF Tissue Cultures 18 Table of Contents (Continued) Page Mosquito Colonies . 18 Mosquito Infection .. 19 Transmission 21 Mosquito Organ Culture 22 Electron Microscopy Techniques a. Thin sectioning 23 b. Negative staining 2h c. Immunochemical staining 2h R E S U L T S Viremia Studies a. Rabbits 26 b. Mice 26 Virus Development i n Tissue Culture " 29 a. H.Ep.. 2 c e l l s .' 29 b. Chick embryo fi b r o b l a s t s at 37°C 29 c. Chick embryo fib r o b l a s t s at 29°C 35 Virus Growth i n Whole Mosquitoes a. Injection of Aedes vexans and Aedes canadensis pools 35 b. Aedes vexans and Aedes canadensis f e d on virus-blood mixtures 38 c. Aedes aegypti injected with virus 38 d. Aedes aegypti fed on viremic mice 38 v i Table of Contents (Continued) Page Insect Tissue Culture a. Gut c e l l s from f i r s t instar larvae UO b. Mosquito gut organ culture hO Electron Microscopy a. Negative st a i n using PTA h3 b. Thin sectioning: chick embryo fib r o b l a s t s ^3 c. F e r r i t i n l a b e l l e d H.Ep. 2 c e l l s U-7 d. Mosquito tissue h7 DISCUSSION • 52. LITERATURE CITED 58 v i i L i s t of Tables Table Page I The Bunyamwera group of viruses 7 I I Members of the Bunyamwera supergroup 8 L i s t of Figures Viremia i n New Zealand white rabbits following i n j e c t i o n of Bunyamwera virus intravenously Serological response of rabbits to Bunyamwera virus Viremia i n Swiss mice following intracerebral i n j e c t i o n of Bunyamwera virus A r t i f i c i a l viremia i n Swiss white mice following intravenous i n j e c t i o n of Bunyamwera virus Development of Bunyamwera i n H.Ep. 2 c e l l s Development of Bunyamwera i n chick embryo fib r o b l a s t s at 37°C Virus development i n chick embryo fi b r o b l a s t s at 29°C Virus development i n Aedes vexans and Aedes  canadensis pools following intrathoracic i n j e c t i o n of Bunyamwera virus Virus t i t r e s i n sal i v a r y glands, gut, and leg sampl from Aedes vexans and Aedes canadensis pools follow ing intrathoracic i n j e c t i o n with Bunyamwera virus Virus development i n Aedes aegypti following intrathoracic i n j e c t i o n with Bunyamwera • Bunyamwera development i n sal i v a r y gland, gut and leg samples from Aedes aegypti fed on viremic mice Virus development i n Aedes aegypti gut.organ culture inoculated with Bunyamwera virus Uninfected chick embryo fibrobl a s t ; c e l l (x20,000) V i r u s - l i k e p a r t i c l e s at the c e l l membrane of chick embryo fib r o b l a s t s at the time of inoculation (xl05,000) i x L i s t of Figures (Continued) Figure Page 15 Precursor-like p a r t i c l e s i n chick embryo f i b r o -blasts four hours post i n f e c t i o n (x62,580) k6 16 Budding p a r t i c l e at the surface of a chick embryo fi b r o b l a s t 12 hours after i n f e c t i o n (xll5,000) kG 17 E x t r a c e l l u l a r p a r t i c l e s i n chick embryo f i b r o -blasts two days after inoculation (x79 3 000) 4^-8 18 Virus containing vacuole i n a chick embryo f i b r o -b l a s t two days after i n f e c t i o n (x57,750) U8 19 F e r r i t i n l a b e l l e d virus i n H.Ep. 2 culture 2k hours after i n f e c t i o n (xl05,800) kQ 20 Midgut c e l l s from uninfected Aedes aegypti (x20,000) k9 21 V i r u s - l i k e p a r t i c l e s i n mosquito f a t body c e l l s 12 days after virus ingestion (xk7,000) 51 22 V i r u s - l i k e p a r t i c l e s i n mosquito gut 10 days after v i r u s ingestion (x50, kOO) 51 .X Acknowledgements The author thanks Dr. J. J. R. Campbell, Head of the Department of Microbiology, for granting.this opportunity f o r study and research. The guidance of Dr. D. M. McLean,' Head of the D i v i s i o n of Medical Microbiology, who supervised t h i s work, and the advice of Dr. J. B. Hudson and Dr. J. E. Bismanis on thesis preparation, are greatly appreciated. Gratitude goes to my fiancee, S a l l y Stewart, f o r her help and encouragement. The typing of t h i s manuscript by Miss Rosemary Carter and the technical assistance of Mrs. Teresa Walters are g r a t e f u l l y acknowledged. x i Abbreviations BUN Bunyamwera virus CEF chick embryo fib r o b l a s t s DDSA Dodecenyl succinic anhydride ELY Earle's balanced s a l t solution with lactalbumin hydrolysate and yeast extract GLY Gey's balanced s a l t solution with lactalbumin hydrolysate and yeast extract HBSS Hanks' balanced s a l t solution H.Ep.2 Human e p i t h e l i a l ' c e l l l i n e . Derived from carcinoma of the larynx HI hemagglutination i n h i b i t i o n LI) l e t h a l dose k i l l i n g 50$ of individuals tested 50 mg milligram ml m i l l i l i t e r NI neutralization index nm nanometer pH logarithm of the re c i p r o c a l of the hydrogen ion concentration RNA ribonucleic acid rpm revolutions per minute Introduction and Literature Review Arboviruses a) General Characteristics Arboviruses are unique among v i r a l agents i n possessing the a b i l i t y to multiply i n both vertebrate hosts and hematophagous inverte-brate vectors. They are small (20 nm to 100 nm), contain RNA, and t h e i r i n f e c t i v i t y is- inactivated by sodium deoxycholate and by di e t h y l ether, due to disruption of t h e i r lipoprotein envelope. {h,7h) A l l arboviruses are pathogenic for newborn mice. Following intracerebral in j e c t i o n , they induce an acute nonsuppurative encephalitis with p e r i -vascular lymphocytic cuffing, degeneration of neurons and f o c a l areas of inflammation. (k2,k6) Some agents induce systemic manifestations, e.g. h e p a t i t i s due to yellow fever, hemorrhagic fever due to dengue, and mild f e b r i l e i l l n e s s due to Ilesha. I t should be noted that arbo-virus infections are most often s u b c l i n i c a l and a substantial portion of the world's population has at one time or another been so affected. . Natural transmission of arboviruses i s mediated by Culicidae (mosquitoes, especially c u l i c i n e ) , Chironomidae (Culicoides f l i e s ) , Psychodidae (phlebotomus f l i e s ) , and Ixodidae ( t i c k s ) . Hosts may be mammalian, r e p t i l i a n , or avian. b) C l a s s i f i c a t i o n Currently there are 271 catalogued arthropod-borne viruses. ( These are divided into 38 antigenic groups by the hemagglutination and complement f i x a t i o n reactions of Clarke and Casals. (10,25) Members within a group cross react by hemagglutination or complement f i x a t i o n tests. Minor factors which affect c l a s s i f i c a t i o n are circumstances of 2 i s o l a t i o n , effects on tissue culture and laboratory animals. In addition to those that f a l l into the above catagories, there are an additional 30 or more with no proven r e l a t i o n to each other or the major groups. Group A contains over 20 viruses a l l of which have mosquito vectors. Group B, k2, eight of which are transmitted by Ixodid t i c k s . Group C contains over 10 mosquito-borne agents. (12) The other groups of note are Phlebotomus fever and Changuinola which are phlebotomus-borne, C a l i f o r n i a and Bunyamwera, mosquito-borne, and ungrouped agents with t i c k or mosquito vectors. Recently discovered relations between Bunyamwera group viruses and other small groups has led to the creation of the Bunyamwera Supergroup. (78) In general, i n d i v i d u a l serotypes are l o c a l i z e d geographically, except dengue which i s prevalent throughout the Tropical Zone. However, with modern transport and migratory birds, they may be transferred to a new host-vector system a great distance away. Localization may be p a r t i a l l y explained by the complex epidemiology of the host-vector system, e.g. r e l a t i v e avian i n s u s c e p t i b i l i t y to Tensaw virus not only eliminates a natural reservoir, but also prevents long-range migratory spread. (71) c) Infection Cycles Arboviruses are maintained i n nature by i n f e c t i o n cycles. {kT) In the case of mosquito vectors these are of two types. 1 . Transmission from man to man as with epidemic urban yellow fever. (70) 2. Transmission to man from w i l d or domestic vertebrates, including 3 birds, which act as natural reservoirs f o r the disease. The l i n k s i n these cycles may a l l be considered variables which are affected d i f f e r e n t l y by ecological stresses. The effect of each variable on the propagation of the virus i s therefore of the utmost importance. The host and reservoir vertebrates must be susceptible to the virus and capable of maintaining a s u f f i c i e n t l y high viremia to infect arthropod vectors. The virus also effects a s p e c i f i c range of vectors, which have a low enough i n f e c t i o n threshold to allow viremic animals to i n f e c t them. They must also be capable of supporting virus m u l t i p l i c a t i o n so i n f e c t i v e virus may be transmitted to hosts. (16,62) Host ranges may be tested i n the f i e l d by serology and attempts at virus i s o l a t i o n , and i n the laboratory s u s c e p t i b i l i t y may be tested by inoculation or arthropod-mediated inf e c t i o n . (6) d) Replication of Arboviruses i n Mosquitoes The dynamics of virus-mosquito i n t e r r e l a t i o n s have been of interest since Reed implicated the mosquito as the vector for the "parasite of Yellow Fever" i n 1901. (58) He noted that after a meal from a c l i n i c a l case of yellow fever there was a twelve day latent period before a mosquito could transmit the disease. Stokes, 27 years l a t e r , noted that the agent involved was f i l t r a b l e , transmitted to monkeys by mosquitoes, infected mosquitoes for l i f e , but was not transmitted t r a n s o v a r i a l l y from adult to larvae. (70) Whitman showed yellow fever t i t r e s i n mosquitoes declined f o r several days after a meal, only to r i s e after a week to a l e v e l much higher than that of k the inoculum. (77) Evidence of similar invertebrate m u l t i p l i c a t i o n with other arboviruses showed direct correlation between insect virus t i t r e s , the period of eclipse, and the infectious period which follows a dynamic increase i n virus l e vels. ( l h , h 3 ) During t h i s period similar dynamics were discovered i n the analogous plant virus-insect vector systems. These plant viruses also show cases of transovarial virus passage, a factor which has been a subject of controversy but never proven i n the case of arboviruses. (8,k8) While arboviruses multiply w e l l within mammalian temperature ranges t h e i r growth i n arthropods i s quite slow and dependent on the insect's environmental temperature. The optimum i s generally considered to be 80°F. With higher tempera-tures, the ingested virus rapidly goes into the eclipse phase and the time p r i o r to transmission i s shortened, but mosquito v i a b i l i t y i s decreased and peak virus t i t r e s are si m i l a r to 80°F incubation. The net res u l t i s a decreased i n f e c t i v i t y span. Low temperatures res u l t i n prolonged virus incubation and p o t e n t i a l l y lower t i t r e s . ( 1 5 ) Increa-sing inoculum also decreases e x t r i n s i c incubation time as w e l l as insuring a higher i n f e c t i o n rate. Within mosquitoes, i n i t i a l m u l t i p l i c a t i o n of virus occurs i n the abdominal part of the midgut. The gut i t s e l f shows a degree of s p e c i f i c i t y i n the range' of viruses i t can support, and therefore can act as a bar r i e r to infection. I f the gut i s by-passed by intra-thoracic i n f e c t i o n or by gut puncture the insect can support growth of agents which are outside of i t s natural range.(kk) From the gut, dissemina-5 t i o n takes place v i a the hemolymph to a l l parts of the body. The area of major importance i s the sal i v a r y glands as the mosquito injects s a l i v a containing digestive enzymes and anticoagulents subcutaneously p r i o r to taking a blood meal. (56) Virus multiplying i n the sal i v a r y glands i s shed into the sa l i v a ; and with i n f e c t i o n transmission to the new host i s accomplished. The greatest i n f e c t i v i t y per unit weight i s i n the salivary glands with virus levels of over 6 logs chick LD.-_ being 50 reported. (75) I t should be noted that while most arboviruses show no pathological effect on t h e i r vectors, Semliki Forest produced sa l i v a r y membrane degeneration and decreased secretion. (1+9) e) Electron Microscopy of Replication The electron microscope has proven to be a valuable t o o l i n arbovirology within recent years. Early work was confined to general morphology and correlation of virus sizes with information gained from f i l t r a t i o n and centrifugation experiments.(6k) With the advent of thi n sectioning, electron microscopy made i t possible to delve further into the histo and cytopathology of arbovirus infection. Morgan studied WEE (Western equine encephalomyelitis) development, observing 22nm spheres, which he considered precursors, d i f f e r e n t i a t i n g at membranes, passing through them, and i n the process becoming mature enveloped p a r t i c l e s of 1+5 - 1+8 nm. (50) Group A viruses develop by the enveloping of nucleoids which bud through the plasma membrane. Vacuoles are formed i n the cytoplasm but, while r e l a t i v e l y few p a r t i c l e s are found within them, v i r a l RNA and protein synthesis occurs on t h e i r membranes. As c e l l s degenerate 6 nucleoids are seen around these vacuoles and buds may be seen within those which are not encircled by nucleoids. Group A p a r t i c l e s average 55 nm i n size. (2,22,29) Group B p a r t i c l e s are smaller at 38 nm. They tend to bud at i n t r a - c e l l u l a r membranes and c o l l e c t within distended vacuoles. These vacuoles then pass to the c e l l ' s surface to discharge the v i r i o n s . (1,57) Arbovirus m u l t i p l i c a t i o n i n insect tissue has only recently been confirmed by electron microscopy. Blue tongue virus was tenta-t i v e l y i d e n t i f i e d i n the sal i v a r y glands of Culicoides f l i e s i n 1966. Both enveloped and non-enveloped v i r i o n s were seen.(9) F i l s h i e and Rehacek discovered that group B viruses behaved s i m i l a r l y i n mosquito c e l l cultures and mammalian c e l l l ines when theyobserved JBE (Japanese B encephalitis) and MVE (Murray Valley Encephalitis) maturing at i n t r a -c e l l u l a r membranes surrounding vacuoles. (27) Although Bergold (7) f e l t he had discovered yellow fever p a r t i c l e s i n Aedes aegypti s a l i v a r y glands i n 1962, the f i r s t t r u l y positive findings on tissue from infected mosquitoes were made by Janzen i n 1970 which Chikungunya virus was visua-l i z e d i n Aedes aegypti s a l i v a r y glands. Precursor p a r t i c l e s (size 25 - 31 nm) were seen not only i n the cytoplasm and on vesicles, but also within the nucleus. P a r t i c l e s with diameters 50 - 58 nm were seen within vacuoles and i n the ext r a c e l l u l a r spaces. The only v a r i a t i o n from normal function i s an increase i n the density of sal i v a r y secre-tions. (36) Eastern Equine Encephalomyelitis i n mosquito sa l i v a r y Table I: The Bunyamwera Group of Arboviruses (47,12) Virus Distribution Batai Palearctic, Oriental W.H.O. I 9 6 1 . W.H.O. Techn. Rep. Ser. No. 219 Bunyamwera Ethiopian Smithburn et a l . 19U6. Amer. J. Trop. Med., 26: Cache Valley Nearctice, Neotropical Holden and Hess. 1959. Science 130: II87 Germiston Ethiopian Kokernot et a l . i960. Amer.. J. Trop. Med. 9_: 62 Guaroa Neotropical Groot et a l . 1959. Amer. J. Trop. Med. 8: 6oh Ilesha Ethiopian Okuno. 1961. Amer. J. Trop. Med. 10: 223 K a i r i Neotropical Anderson et a l . i960. Amer. J. Trop. Med. 9: 70 Lokern Nearctic Catalogue of Arthropod-borne viruses, No. 220 Maguari Neotropical Causey et a l . 1961. Amer. J. Trop. Med. 10: 227 Main Drain Nearctic Catalogue of Arthropod-borne viruses,. No. 219 Shokwe Ethiopian Sororoca Neotropical W.H.O. I967. W.H.O. Techn. Rep. No. 369 Tensaw Nearctic W.H.O. I967. W.H.O. Techn. Rep. No. 369 Wyeomyia Neotropical Roca-Garcia. 19M+. J. Infect. Dis. 75: 160 Table I I : Members of the Bunyamwera Supergroup of Viruses Group No. i n Group Bunyamwera Ik Group C 11 Guama 8 Capim 7 Simbu 16 Bwamba 2 C a l i f o r n i a 10 Patois k Tete 3 Koongol 2 O l i f a n t v l e i 2 Unassigned k 9 glands shows p a r t i c l e s i d e n t i c a l to those seen i n mammalian c e l l s . Wo nuclear virus i s seen, and random budding from c e l l membrane systems seems to be the method of release. Virus and salivary secretions accumulate at the apical region of the c e l l , passing from here into the sal i v a r y ducts. Large numbers of v i r i o n s are not seen i n the cytoplasm. (76) The low rate of production and r e l a t i v e lack of overt pathology explains how arboviruses manage to confer l i f e long i n f e c t i v i t y on a mosquito without affecting i t s l i f e span. General Characteristics of Bunyamwera and the Bunyamwera Group • a) C l a s s i f i c a t i o n Bunyamwera i s the prototype s t r a i n of a group containing Ik agents (see Table I) which show serologic cross reaction by complement f i x a t i o n and hemagglutination i n h i b i t i o n tests. I n i t i a l l y these cross reactions were f e l t to be s t r i c t l y within the group, but more recent study has shown i n t e r r e l a t i o n with some other pre-established groups. (2.1,2k) The r e s u l t i n g aggregation i s the Bunyamwera supergroup com-posed of 11 groups plus unassigned viruses.(78) (See Table II.) The antigenic l i n k s within the supergroup are not necessarily very strong, and not a l l members cross react. b) History Bunyamwera was f i r s t isolated by Smithburn i n 19^ -3 from a large pool of Aedes mosquitoes collected at Bunyamwera, an uninhabited area of the Semliki Forest of Uganda. A monkey which had received a subcutaneous i n j e c t i o n of pooled mosquitoes developed severe symptoms at kk hrs. A post mortem l i v e r sample was injected i n t r a c e r e b r a l ^ . into mice which developed encephalitis and died. (66) Further work showed i t to be an arbovirus sensitive to ether and sodium desoxy-cholate of size 70 - 105 nm by f i l t r a t i o n . (67,73) I t was re-isolated from mosquitoes i n Tongaland i n 1955 i n suckling and weaned mice. The in f e c t i o n rate of Bunyamwera and R i f t Valley i n mosquitoes i n t h i s area was found to be 0.2 - 0.5$. (37) The c l i n i c a l picture of Bunyam-wera i n f e c t i o n was brought to l i g h t by c l i n i c a l investigations of the agent's oncolytic properties. (kO) No b e n e f i c i a l therapeutic effect was discovered, but one subject developed a near-fatal encepha-l i t i s with severe b i f r o n t a l headache, marked fever, h y p e r e x c i t a b i l i t y and laboratory findings indicative of v i r a l encephalitis. This pro-gressed to coma. On subsequent recovery the patient showed decreased mental function and marked amnesia. (68) A young mosquito co l l e c t o r i n Tongaland became the f i r s t recognized case of naturally acquired Bunyamwera. His serum provided the f i r s t virus isolated from a natural human infe c t i o n . This case presented with mild fever, headache, s t i f f neck, but l i t t l e incapacitation. The symptoms lasted two days, after which a r i s e i n anti-Bunyamwera antibody could be detected by neutralization and hemagglutination i n h i b i t i o n tests. Two mosquito virus i s o l a t i o n s were made on the same day, i n the same area where the boy was working. (38) Ukauwa, Ilesha and Germiston were also isol a t e d i n A f r i c a during t h i s period and were promptly c l a s s i f i e d as members of the Bunyamwera group. (5^) Accidental laboratory infections with Germiston gave pictures similar to, but less severethan those reported for Bunyam-wera. (39) Calovo, Ilesha, Guaroa and Ukauwa have also been associated with c l i n i c a l disease. (V7) Two members of t h i s group are endemic i n North America. F i r s t i s o l a t e d was Cache Valley from mosquito pools collected i n Utah i n 1956. (30) Subsequent studies showed a d i s t r i b u t i o n that extended as f a r as B r a z i l . (13) In 1961 Tensaw was i s o l a t e d i n mosquito samples taken from Arkansas. The virus i s 70 - 122 nm i n size by f i l t r a t i o n , shows widespread d i s t r i b u t i o n i n Alabama, F l o r i d a and Georgia, and fu r t h e r - i s o l a t i o n s have been made from mosquitoes and a dog. (18,19, 20,21) . Suckling mice appear to be the only animal i n which Tensaw. gives overt i n f e c t i o n . Dogs show a transient viremia and mcs quito trans -mission from dog to mouse has been accomplished. (71) c) Eeplication of Bunyamwera I n t r a c e l l u l a r r e p l i c a t i o n of Bunyamwera group was f i r s t studied with Guaroa i n a mammalian tissue culture system. Using f l o u r -escent antibody, acridine orange and electron microscope, virus r e p l i -cation was shown to be s t r i c t l y cytoplasmic with diffuse areas of ENA production and scattered, descrete f o c i of v i r a l antigen production. (69) The histopathology of f a t a l Bunyamwera encephalitis i n suckling mouse brain shows i n t e r s t i t i a l and perivascular edema, neuronal necrosis, a lack of inflamatory response (due to the animal's immaturity) and' no i n c l u s i o n bodies. Cytopathology f i r s t shows at 20 hrs. with the occa-sional appearance of v i r i o n s . By 3^+ hrs. virus i s present i n the e x t r a c e l l u l a r spaces, Golgi and endoplasmic reticulum membranes increase i n quantity, and there are signs of mitochondrial degeneration. The nucleus appears undisturbed. Virus t i t r e s within the c e l l are very high p r i o r to the outset of marked degenerative change. Virus buds into cisternae, attached to the membrane by a stalk which pinches off. Accumulations of nucleoids are not seen. Elongated, s t r i a t e d p a r t i c l e s of the same diameter (98 nm) as round v i r i o n s are also present. In l a t e stages large vacuoles distended with p a r t i c l e s , and small vacuoles with i n d i v i d u a l viruses, are seen. I t i s f e l t that virus release i s by c e l l disruption and/or fusion of the small vacuoles' membranes with the plasma membrane followed by virus egestion. This mechanism and morphology seems to be common to a l l of the Bunyamwera and C a l i f o r n i a group viruses.(51,52) Ukauwa virus m u l t i p l i e s i n A. aegypti after feeding and i n j e c t i o n and i n A. canadensis and A. t r i s e r i a t u s following intrathora-c i c inoculation. In A. aegypti an e x t r i n s i c incubation period of 8 days was noted following an infectious meal. Peak t i t r e s of k.3 log mouse L DJ - Q i n the thorax, 5.3 log mouse L C ^ Q i n the s a l i v a r y glands, h.3 log mouse L B ^ Q i n the legs and k.6 log mouse L D ^ Q i n the gut were reached on day ik after the infectious meal. With parenteral inoculation i n f e c t i v i t y was detected within 12 hrs. and had peaked by k days. Virus t i t r e s were s l i g h t l y higher by t h i s method, the highest levels being found i n the thorax. Virus transmission to mice was possible hrs. after i n f e c t i v i t y showed i n the sa l i v a r y glands, provided that threshold t i t r e of at least k.3 log mouse L L \ - Q was present. (53) A comparative study of Bunyamwera virus reproduction i n vertebrate and insect tissues i s the objective of t h i s thesis. Materials and Methods Materials and Methods Virus The prototype s t r a i n of Bunyamwera virus from the American Type Culture Collection was obtained i n i t s f o r t y - s i x t h suckling mouse brain passage.(66) A stock virus preparation was prepared by inocu-l a t i o n of a family of 10 suckling mice aged 2 days with 10^ mouse L D ^ Q -Brains were removed a s e p t i c a l l y from mice two days l a t e r when signs of encephalitis were observed. The brains were homogenized using a mor-tar and pestle and suspended i n ELY plus 20$ c a l f serum, centrifuged at 2000 rpm for 3 minutes to remove gross tissue debris, and stored at -70°C i n 0.1 ml. aliquots i n sealed glass ampuoles. Virus Assay Virus assays were carried out by intracerebral inoculation with 0.03 ml. of s e r i a l tenfold dilutions of test material into groups comprising 3 Swiss mice each aged 3 weeks. Virus caused f a t a l encepha-l i t i s within 5 days. Virus dilutions were made i n ELY or s t e r i l e 0.15 M sodium chloride plus 20$ c a l f serum. The mouse LDr„ t i t r e s were calcu-50 lated by the method of Karber. (k-T) Blood samples were dilut e d 1:2 on c o l l e c t i o n with Alsever's solution. Mosquito organs and whole mosquitoes were ground i n a mortar with 0.9 ml. of s t e r i l e 0.15 M sodium chloride containing 20$ c a l f serum. Tissue culture scrapings suspended i n ELY + 20$ c a l f serum and tissue culture supernates were also used as s t a r t i n g suspensions. Serology Neutralization tests were carried out by mixing 0.15 ml. of undiluted anti-Bunyamwera rabbit serum with 0.15 KLL. of s e r i a l tenfold dilutions of virus. (^5) These samples, along with a s e r i a l tenfold d i l u t i o n virus control, were held at 25°C for 30 minutes prior to intracerebral i n j e c t i o n into groups of weaned mice. The virus control determined the number of L D , - ~ of virus actually used i n the test. Mice - 50 receiving unneutralized virus developed f a t a l encephalitis within f i v e days. The log^Q neutralization index, was the difference between the control virus t i t r e and that of the.serum-virus mixture. Hemagglutination i n h i b i t i o n antibodies were measured by the method of Clarke and Casals (25) using disposable M i c r o t i t r e plates. (63) S e r i a l two f o l d dilutions of 1:5 serum suspensions which had been acetone extracted were tested against 8 hemagglutinating doses of sucrose-acetone extracted, Bunyamwera infected mouse brain antigen. Preparation of Anti-Bunyamwera Serum A 10 mouse L D ^ g suspension of Bunyamwera i n 1 ml. of 20$ c a l f serum saline was injected into the marginal ear vein of two New Zealand white rabbits. Serum samples were "taken at selected intervals' to assess the production of antibody. A second dose of 10^ mouse L B ^ Q was injected i n the marginal ear vein k weeks l a t e r . One week after t h i s booster i n j e c t i o n 20 ml. of blood was collected from.an ear vein. Following centrifugation at 2000 rpm to remove the c l o t , the serum was removed and stored at -20°C i n screw cap v i a l s . Viremia Experiments a) Rabbits Blood samples were drawn from ear veins at 0, 2, 3, k, 6, and 7 5 8 hours after the i n j e c t i o n of 10 mouse LD„ into the marginal ear 50 vein of New Zealand white rabbits. These samples were stored i n Alsever's solution at -20°C.(23) b) Mice 1. Intraperitoneal inoculation. Three 3 week o l d mice each each of 10 LD^^ml. of Bunyamwera intraperitoneally. Blood was received 0.1 ml. of 1 0 U * J J LD^o/ml. and another three received 0.1 ml. removed from the t a i l vein and by cardiac puncture when symptoms of encephalitis were seen. This was placed i n Alsever's solution and stored at -70°C p r i o r to testing. 2. Intracerebral inoculation. Six three week old mice received k 8 10 * mouse LD,__ intra c e r e b r a l l y i n 0.03 ml. each of a Bunyamwera sus-50 pension. They were bled from the t a i l vein at d a i l y intervals u n t i l they died from encephalitis. These blood samples were stored i n Alsever's solution at -70°C u n t i l used. 3. A r t i f i c i a l viremia. Mice were injected i n the t a i l vein 7 5 with 0.5 ml. of a 10 ID^/ml. Bunyamwera suspension i n a method analogous to that used by Chernesky f o r creating a r t i f i c i a l viremias i n rabbits with feeding t i c k s . (23) Blood samples were taken at the time of infe c t i o n , 30 minutes, 1 hour, 2 hours l a t e r , and then tested f o r virus t i t r e s . Tissue Culture Infected vertebrate tissue cultures were used as a comparison for insect tissue v i r u s growth. H.Ep. 2, mouse macrophage, and primary chick embryo f i b r o b l a s t (CEF) cultures were t r i e d . CEF was found to be most satisfactory i n terms of virus production and convenience. CEF cultures were produced by a s e p t i c a l l y removing embryos from eggs which had been incubated at 37°C for 10 to 12 days. The limbs, eyes, and beak were removed. The remaining tissue was placed i n a s t e r i l e p e t r i plate and f i n e l y minced with scissors". Hanks' balanced s a l t solution +0.25$ try p s i n was added, and the mixture agitated on a magnetic s t i r r e r f o r f i v e minutes at room temperature. This supernate was discarded and the tissue transferred to a t r y p s i n i -zing f l a s k . The washing was repeated, t h i s supernate also being d i s -carded. Eight ml. of 0.25$ trypsin-HBSS was added per embryo and s t i r r e d f o r 30 min. at 25°C. The supernate- was pipetted into a ho ml. centrifuge tube containing 1.5 ml. of c a l f serum. This extraction was repeated three times. The c e l l s were deposited by centrifugation at 1500 rpm f o r 10 minutes, and the supernate was discarded. The upper part of the packed c e l l s which was free of erythrocytes, was removed and suspended i n GLY. The c e l l concentration of t h i s suspension was estimated by d i l u t i n g a sample 10 i n 0.1 M c i t r i c acid and 0.1$ c r y s t a l v i o l e t and counting i n a hemocytometer. The concentration was adjusted to 2 x 10^ cells/ml. using GLY plus 5$ ca l f serum. The res u l t i n g suspension was dispensed i n 5 ml. amounts into 60 mm. p l a s t i c p e t r i plates which were incubated i n small, sealed battery jars at 37°C u n t i l the c e l l s formed monolayers.(kl) Infection of CEF Tissue Cultures After the tissue culture media had been decanted, the c e l l s were covered with 1 ml. of GLY-5$ c a l f serum virus suspension contain-5 2 o ing 10 " mouse LI) of Bunyamwera per ml. After 30 minutes at 37 C 50 the inoculum was removed and replaced with 5 ml- of GLY plus 5$ ca l f serum maintenance medium, the plates were then incubated i n sealed small battery jars at 29°C or 37°C. These were checked d a i l y f o r cytopathic effect using an inverted stage l i g h t microscope. Virus t i t r e s were measured at 0, k, 8, and 12 hours, and 1, 2, k, and 10 days. C e l l samples were removed for electron microscopy at the same i n t e r -vals. Mosquito Colonies a) Adult Female Aedes aegypti mosquitoes were provided from the colony at the B r i t i s h Columbia Research Council Laboratories at the University of B r i t i s h Columbia. These were kept i n cardboard and nylon mesh con-tainers within a b e l l jar at 80$ humidity and 25°C. Maintenance feed-ing was accomplished by placing s p l i t r a i s i n s or cotton wool b a l l s 19 soaked i n a sucrose-water solution on the cage tops. b) Aedes aegypti eggs were also provided by the B r i t i s h Columbia Research Council Laboratory. These were hatched by placing them i n a deionized water plus yeast extract solution i n a pan within a nylon mesh holding cage. When pupae matured into adults they were removed to small cardboard and nylon cages and kept as i n a. c) Wild Aedes vexans and Aedes canadensis collected by hand at sit e s around Penticton, B r i t i s h Columbia were a i r freighted to Vancouver i n small cages within a tape-sealed styrofoam cooler. These were then transferred to small holding cages and maintained as i n a. These two species were pooled f o r experiments. Mosquito Infection a) Inoculation Quarter inch Pyrex tubing was heated over a bunsen burner flame and drawn out t i l l i t had a diameter of approximately 10 u. This was attached to a 0.25 ml. syringe by a rubber tube coupling. The plunger was operated by a low p i t c h screw which minimized volume va r i a t i o n i n s e r i a l injections. By weighing volumes expressed from the syringe i t was found that one quarter turn of the screw delivered 1 mg. (.001 ml.) of the inoculum. The virus stock was suspended i n s t e r i l e saline plus 10$ c a l f serum and pulled into the syringe. Single mosquitoes were aspirated from the holding cage and placed into a three inch length of one inch glass tubing which had one end covered with nylon mesh and the other stoppered with cotton wool. Carbon dioxide fumes from a tank or a bottle .of dry ice were directed into t h i s chamber to anaesthetize the mosquito, which was then tipped onto the stage of a dissecting microscope. The syringe's capillary-tube was inserted through the cuticle.of the thorax midway between the base of the wing and base of the hind leg and the screw was given a 3/4 turn. The insects were then placed i n holding cages i n the insectary at 25°C u n t i l they were sampled. b) A sugar cube was placed on the mesh top of a cage of mosquitoes which had been starved f o r one day. A measured d i l u t i o n of virus i n defibrinated rabbit blood was pipetted onto the cube. The mosquitoes which fed within three hours and were engorged were removed to a hold-ing cage at 25° to await sampling. The t i t r e of virus on the cube was measured before and after feeding. , c) An adult Swiss white mouse was secured to a table with adhesive tape, l i g h t l y anaesthetized with ether, and i t s abdomen shaved. A 26 gauge needle was inserted into the t a i l ' v e i n and 0.5 ml. of virus i n saline-10$ c a l f serum was injected. A mosquito cage was immediately secured over the mouse i n a manner which would allow the insects to feed on the. mouse through the cage's mesh. Blood samples f o r viremia tests were taken from the t a i l vein at the time of i n j e c t i o n and at the end of feeding (30 minutes). Engorged mosquitoes were removed and placed i n a holding cage at 25°C. In a l l cases a sample pool was taken immediately after feed-ing (0 hours) to t i t r a t e the i n f e c t i v i t y of the meal. Samples f o r v i r u s t i t r a t i o n and electron microscopy -were taken at 0, 2, and 12 hours, and 1, 2, 3, h, and 10 days. Pools of three mosquitoes were anaesthetized by placing them i n -h°C for f i v e minutes and dissected i n s t e r i l e saline with 20$ c a l f serum under a dissecting microscope. The wings were removed using a s t e r i l e scalpel and discarded. The legs were cut off using a new s t e r i l e blade and pooled i n a mortar for t i t r a t i o n . Salivary glands were expressed by t r a c t i o n on the head of the mosquito while applying gentle pressure to the thorax. These were divided with a s t e r i l e blade -- h a l f f o r electron microscopy and h a l f pooled i n a mortar f o r t i t r a t i o n . The gut was removed by nicking the w a l l of the abdomen at the l a s t segment and extracting the gut by gentle t r a c t i o n on the separating segment. The midgut was cut away and halved f o r t i t r a t i o n and electron microscope procedures. Material f o r t i t r a t i o n was placed i n screw cap v i a l s and stored at -70°C. Transmission Suckling mice were secured to a bench top with tape and cages containing i n d i v i d u a l infected mosquitoes were placed over them. Engorged mosquitoes were harvested f o r t i t r a t i o n and electron micros-copy after two hours and mice that had been b i t t e n were returned to t h e i r mother. The c r i t e r i o n f o r transmission of i n f e c t i o n was deve-lopment of signs of encephalitis within f i v e days of being b i t t e n . The harvested brains of these moribound mice were sampled for i n f e c t i -v i t y . 22 Mosquito Organ Culture Primary mosquito gut cultures were produced to provide a convenient and e a s i l y controllable arthropod tissue environment for virus r e p l i c a t i o n . Tissues were grown i n Leighton tubes at 28°C using Grace's insect tissue culture medium (Grand Island B i o l o g i c a l Co.) plus 5$ c a l f serum i n place of insect hemolymph. (28) a) Gut tissue was obtained from s t e r i l e f i r s t instar larvae of Aedes  aegypti. Eggs were l a i d on damp s t r i p s of f i l t e r paper. These were allowed to dry and then were washed by dipping the s t r i p s into 95$ ethanol, drying them i n a s t e r i l e p e t r i plate and repeating the proce-dure two more times. The eggs were hatched i n a s t e r i l e 0.5$ yeast extract-water solution. The anterior thirds of the larvae were cut off using a s t e r i l e scalpel blade and discarded. The remainder was diced with a s t e r i l e scalpel blade, suspended i n 1 ml. growth medium per f i v e larvae, dispensed i n 1 ml. amounts into Leighton tubes and incubated at 28°C u n t i l c e l l attachment was seen. The c e l l s are f i b r o -b l a s t - l i k e and grow from the ends of tissue fragments. Degeneration of the c e l l s took place within one week after culturing. b) Adult Aedes aegypti were fed on mice. The distended midguts were removed and placed into Grace's insect tissue culture medium along with a n t i b i o t i c s to supress b a c t e r i a l and yeast growth. Two guts i n 1 ml. of medium were used per tube which was then placed i n a 29°C incubator. These preparations were viable f o r 10 to 12 days. Electron Microscopy Techniques Electron microscopy was used to study the cytopathology of Bunyamwera i n both mosquito and chick embryo tissues. (55) a) Thin sectioning Tissue culture specimens were scraped from the glass using a rubber policeman, pelleted by centrifugation and immediately placed into 2.5$ glutaraldehyde i n 0.1 M phosphate buffer pH 7-2 for 30 minute at k°C. This was given three 1 hour washes i n phosphate buffered sucrose (0.2 M) at k°C. Tissues were post f i x e d i n 1$ OsO^ pH 7.2 phosphate buffer at k°C. The f i x a t i v e was aspirated and the sample was given three 15 minute washes i n k°C d i s t i l l e d water. Dehydration was carried out by passing the specimen through a graded series of ethanol concentrations: 15 minutes i n 30$; 5 minutes each i n 50$> 75$? and 95$5 and three 5 minute changes of absolute ethanol. Samples were embedded i n Epon 812 resin using DDSA and methylnadic anhydride as curing agents. The procedure involved two 15 minute changes of propylene oxide, a one hour change of Epon-propylene oxide 1:1 and one hour of Epon alone. The samples were trans ferred to Beem capsules which were f i l l e d with Epon, incubated at 37° C for 12 hours to f a c i l i t a t e r e s i n penetration, and cured at 60° for 36 hours. Mosquito samples were treated i n the same manner as tissue cultures except a l l times i n the f i x a t i o n and dehydration procedures were doubled to counteract poorer tissue permeability. . The hardened blocks were cut using a LKB I I I microtome and a glass knife. The sections were collected on carbon coated formvar films on 1+00 mesh copper grids. These were p o s i t i v e l y stained with a satur-ated alcoholic solution of uranyl acetate f o r 1 minute, washed with d i s t i l l e d water f o r f i v e minutes, and then post-stained with Reynold's lead c i t r a t e at room temperature for 10 to 15 minutes. (60) Grids were washed with d i s t i l l e d water and a i r dried p r i o r to examination with a P h i l l i p s EM 300 electron microscope. b) Negative Staining The c e l l s of an. infected mouse brain preparation were d i s -rupted by rapid freeze-thawing. The sample was placed on a metal plancet which was a l t e r n a t i v e l y touched to a block of dry ice and the palm of a hand. The c e l l clumps were pelleted by low speed c e n t r i f u -gation and the p e l l e t s resuspended i n d i s t i l l e d water. A drop of t h i s suspension was mixed with a drop of 3$ PTA (phosphotungstic acid) pH 6.5 on a 300 mesh carbon and formvar coated copper grid. The excess l i q u i d was removed by touching the edge of the g r i d with f i l t e r paper. The g r i d was a i r dried and observed i n a P h i l l i p s EM 300 electron microscope. c) Immunochemical Staining f o r Electron Microscopy Rabbit anti-Bunyamwera serum was treated with 1.4-36 M sodium sulfate to i s o l a t e the globulin f r a c t i o n . This f r a c t i o n was conjugated with six-time r e c r y s t a l l i z e d cadmium-free f e r r i t i n by the method of Ri f k i n d e t _ a l u t i l i z i n g toluene 2, k- diisocyanate. (61,65) Tissue samples were f i x e d i n phosphate buffered formalin 5$ at pH 7.2 for 10 minutes, then quick frozen i n an alcohol-CO^ bath and thick sectioned on a cryostat. These sections were stained with the conjugate, then washed, f i x e d with phosphate buffered osmium t e t r o -xide IPJo at pH 7.2 and embedded for electron microscopy. Results 26 Results Viremia Studies a) Rabbits 7 5 Two rabbits received 10 mouse L D ^ Q of Bunyamwera (BUN) virus i n 1.0 ml. amounts. Average viremia t i t r e s were 10 mouse 3 1 L D J - Q per ml. at zero hours decreasing to 10 mouse L D ^ Q per ml. at four hours, and by eight hours virus became undetectable (Fig. l ) . Virus was cleared at a rate of 10^*^ mouse L D , _ _ per ml. per hour. 50 Serum samples taken p r i o r to inoculation did not possess any anti-BUN antibodies. Neutralizing antibody f i r s t appeared at nine days post i n j e c t i o n at a neutralizing index of 2.5 mouse L L V „ 50 per ml. of serum. By 25 days t h i s had r i s e n to 4.0 mouse L I ) per 50 ml. of serum. Booster injections of 10 mouse L D ^ ^ given at eight weeks were followed by an antibody t i t r e of 5*4 mouse L D , - , . n e u t r a l i -50 zing doses per ml. one week l a t e r (Fig. 2). Control virus incubation at 25°C with non-immune serum showed an i n f e c t i v i t y loss of l O 1 " ^ mouse L D , _ Q per ml. per hour fo r the f i r s t three hours. Anti-hemagglutinin was f i r s t detected seven days after i n f e c t i o n at a t i t r e of 20 and increased to a maximum of 4-00 seven days after the second i n j e c t i o n (Fig. 2). b) Mice 1. After Intracerebral and Intraperitoneal Infection 5 0 2 4 6 8 h o u r s F i g . 1 . Viremia i n New Zealand white rabbits following i n j e c t i o n of lO?-5 mouse LD50 per ml. of Bunyamwera virus intravenously. r e c i p r o c a l o f h i g h e s t s e r u m d i l u t i o n 10 0 % H I p o s a t i v e »_i ^ 05 N> 00 CD C\3 ^ O O O O O O M CO ^ 01 Oi n e u t r a l i z a t i o n i n d e x Fig. 2. Serological response of rabbits to Bunyamwera virus following intravenous i n j e c t i o n of 1 0 ? • 5 mouse L D ^ Q of virus as tested by hemagglutination i n h i b i t i o n (©) and virus n e u t r a l i z a t i o n ( O ) . 5 3 Intraperitoneal i n j e c t i o n of 10 * ^ D ^ Q virus resulted 2 0 i n a viremia of 10 " mouse L D ^ Q per ml. at three days. After i n t r a -nt-. 8 cerebral i n j e c t i o n of 10 * L D ^ of virus the peak t i t r e of viremia, k 3 10 ' mouse L D ^ Q per ml., was attained at four days, by which time the mice were moribund (Fig. 3). 2. A r t i f i c i a l viremia 7 7 Mice were inoculated intravenously with 10 * mouse L D , - 0 of 50 6 3 Bunyamwera. Blood samples taken at the time of in f e c t i o n showed 10 mouse L D , _ 0 per ml. Mosquitoes were fed on the mice. Another sample 50 taken at 30 minutes post infection, showed a viremia of 10 mouse L D 5.6 r r n n i v M i . i i 50 per ml. of blood (Fig. 4). During the f i r s t two hours post i n f e c t i o n • 2 0 virus was cleared at a rate of 10 ' mouse L D , _ Q per ml. per hour. Virus Development i n Tissue Culture a) H.Ep. 2 c e l l s 6 5 H.Ep. 2 monolayers were infected with 10 mouse LD,_^  of 50 BUR vi r u s . Following a steady decline of I n f e c t i v i t y to undetectable levels at four hours, i n i t i a l evidence of virus production was observed 6 8 after 12 hours. The peak virus t i t r e (10 * mouse L D ^ Q P E R ml. of c e l l -supernate .suspension) was attained at two days following which t i t r e s declined due to complete destruction (Fig. 5). b) Chick Embryo Fibroblasts at 37°C Within two hours after inoculation of CEF monolayers, virus 5 2 t i t r e s declined to zero from an i n i t i a l 10 mouse LD,-~ per ml. There-50 6 2 after the virus t i t r e increased to a peak of 10 * mouse LD,__ at 24 50 hours (Fig. 6). No virus was recovered at four days, by which time c e l l destruction was complete. 0 1 2 3 4 d a y s F i g . -3. Viremia i n Swiss white mice following intracerebral i n j e c t i o n of lCr**" mouse LDj-p. of Bunyamwera v i r u s . 31 Fig. h. A r t i f i c i a l viremia i n Swiss white mice following intravenous i n j e c t i o n of mouse LD of Bunyamwera virus. J I L 0 1 2 3 4 d ay s Fig. 5. Development of Bunyamwera virus i n H.Ep. 2 c e l l s following i n f e c t i o n with 10"' 5 mouse LD(-A per ml. J i L 0 1 2 3 4 d a y s Fig. 6. Development of Bunyamwera virus i n chick embryo f i b r o b l a s t s following inoculation with mouse LDc;o per ml. and incubation at 37°C. O 2 4 6 8 10 d a y s Fig. 7. Virus development i n chick embryo fi b r o b l a s t s infected with 103-7 mouse LDCJQ per ml. of Bunyamwera virus and incubated at 29°C. c) Chick Embryo Fibroblasts at 29°C 3 7 The virus t i t r e declined from an i n i t i a l 10 mouse L D „ 50 2 7 S O to 10 ' at 12 hours, followed by an increase to 10 at four days k 0 and subsequent decrease to 10 * at 10 days (Fig. 7). At that time complete destruction was observed i n 90% of c e l l s . Virus Growth i n Whole Mosquitoes Mosquitoes were injected with a blood-virus mixture contain-7 ing 10 mouse L D ^ Q per ml. of virus. Pools t i t r a t e d immediately after 5 6 inf e c t i o n contained 10 mouse LD,__ per mosquito for the fed insects 50 5 5 and 10 mouse L D ^ Q P E R mosquito f o r injected insects. a) Injection of Aedes vexans and Aedes canadensis Pools The virus content of whole mosquitoes was determined d a i l y 3 2 following intrathoracic i n j e c t i o n of mosquitoes with 10 * mouse ID-.-50 1 h per insect. I n f e c t i v i t y declined to 10 * mouse L D ^ Q & t 1 day, followed by an increase to 10 2'^ at 2 days and a maximum of 10^*^ at 10 days (Fig. 8). After i n j e c t i o n , virus i n f e c t i v i t y was rapi d l y l o s t from the hemolymph, but i n f e c t i v i t y was maintained i n sal i v a r y gland and midgut organ samples. Both gut and salivary tissue demonstrated active virus m u l t i p l i c a t i o n two days post i n j e c t i o n , and virus was 5 2 found i n leg samples. Virus t i t r e s at ten days were 10 mouse ID,.,. 50 kO 2 7 i n s a l i v a r y glands, 10 " mouse LLV^ i n gut and 10 " mouse LD.-„ i n 50 50 leg samples (Fig. 9)-Hydrachnellid mites infect many mosquito species and pass [ 1 I 1 I L O 2 4 6 8 10 d a y s i g . 8. Virus development i n Aedes vexans and Aedes canadensis pools following i n t r a -thoracic i n j e c t i o n of 103•2 mouse L D ^ Q of Bunyamwera v i r u s . 0 1 2 3 4 5 6 7 8 9 10 d a y s F i g . 9- Virus t i t r e s i n s a l i v a r y glands ( O ) , gut (•), and leg O) samples from Aedes vexans and Aedes  canadensis pools following intrathoracic i n j e c t i o n with 10^«2 mouse ID™ of Bunyamwera vi r u s . from insect to insect when mosquitoes are i n close contact. (33,3*0 Mites taken from w i l d mosquitoes at the end of the ten day virus incubation period contained no detectable virus. Therefore, mosquito to mosquito transmission of virus by mites i s not probable. Transmission of virus by b i t i n g suckling mice was accomplished by two of three mosquitoes 10 days after i n j e c t i o n , but not e a r l i e r . 5 2 Thus a sal i v a r y gland virus t i t r e of 10 " correlated with the a b i l i t y of a mosquito to transmit BUN virus to mice. b) Aedes vexans and Aedes canadensis Fed on Virus-Blood Mixtures 6 5 Mosquitoes fed on virus-Blood mixtures containing 10 mouse k 0 L D | - Q per ml. ingested 10 * mouse L D ^ . At eight days the virus levels h. 0 2 8 were 10 * mouse L D ^ Q and declined to 10 * mouse L D ^ Q by 17 days. Virus transmission was not accomplished with these pools. c) Aedes aegypti Injected with Virus 3 3 Mosquitoes injected with 10 mouse L D „ of BUN virus were 50 sampled over a period of four days. An i n i t i a l f a l l i n virus t i t r e ended by 12 hours, by two days virus levels had ri s e n to 10^"^ mouse 5 2 L D , - Q , and at four days the t i t r e was 10 mouse L D ^ Q per mosquito (Fig. 10). d) Aedes aegypti Fed on Viremic Mice Mosquitoes were re a d i l y infected with substantial amounts Fig. 10. Virus development i n Aedes aegypti following intrathoracic i n j e c t i o n with 103- 3 mouse LD^ of Bunyamwera. of virus by feeding on mice with a r t i f i c i a l viremias. Gut levels of k 6 10 * mouse LDcr. were attained at the time of the meal. This f e l l 50 2 6 slowly to 10 * mouse L D , ^ at one day and rose to a peak l e v e l of 50 3 3 10 " mouse L D , _ _ by 10 days. The detection of virus i n the sal i v a r y 50 glands followed the dissemination of virus i n the hemolymph as monitored 5 0 by t i t r a t i o n of legs. Salivary gland levels reached 10 mouse L D ^ ^ by 10 days (Fig. 11). At t h i s time transmission of virus to mice was demonstrated i n both pools tested. Insect Tissue Culture a) Gut Cells from F i r s t Instar Larvae BUN m u l t i p l i c a t i o n occurred at a low l e v e l i n l a r v a l tissue 5 7 cultures. I n i t i a l t i t r e s of 10 mouse L D „ per ml. f e l l to undetec-50 3 0 table levels by one day. The peak t i t r e of 10 mouse LL) per ml. 50 was reached at four days. C e l l u l a r degeneration was evident at this time and by f i v e days v i r t u a l l y a l l tissue fragments were destroyed and had detached from the glass. C e l l death by f i v e days was non-sp e c i f i c because the c e l l s i n the control tube showed a similar cycle. b) Mosquito Gut Organ Culture 3 7 Whole gut cultures infected with BUN virus at 10 * mouse 1 8 LD|_Q per ml. dropped to a t i t r e of 10 * mouse L D ^ per ml. at 12 hours. Rapid growth i n the second 12 hour period brought the t i t r e 5.0 6.0 to 10 mouse L D ^ Q per ml. at one day. Peak t i t r e s of 10 mouse 0 1 2 3 4 5 6 7 8 9 10 d a y s Fig. 11. Bunyamwera vir u s development i n s a l i v a r y gland ( O ) , gut (@), and leg samples (Q) from Aedes aegypti fed on Swiss white mice with a viremia of 10 mouse LD,-n per ml. 0 1 2 3 4 5 6 7 8 9 10 d a y s F i g . 12. Virus development i n Aedes aegypti gut organ culture following inoculation with 1CP'7 mouse I D ^ Q P E R ra^-- °^ Bunyamwera vi r u s . L D ^ Q per ml. were achieved at four days after which they dropped to undetectable levels by ten days. The reason f o r loss of c e l l v i a b i -l i t y i n th i s experiment probably arose from the culturing technique rather than the virus infection, as the control cultures also l o s t v i a b i l i t y by ten days (Fig. 12). Electron Microscopy a) Negative Stain Using Phosphotungstic Acid A suspension of freeze-thawed BUN infected mouse brain was negatively stained and examined i n a P h i l l i p s EM 300 electron micro-scope. In addition to c e l l debris and myelin-like structures numerous p a r t i c l e s with a mean diameter of 100 nm were observed. In cases where the external structure of the p a r t i c l e s had been disrupted a loosely defined inner component of kQ nm mean diameter was seen. I t was not possible to p o s i t i v e l y i d e n t i f y these as virus as st r u c t u r a l d e f i n i -t i o n was--poor. Uninfected mouse brain did not show a similar pattern of 100 nm p a r t i c l e s . b) Thin Sectioning: Chick Embryo Fibroblasts Thin sections of uninfected chick embryo f i b r o b l a s t cultures showed w e l l preserved micro anatomy with intact c e l l membranes, mito-chondria, and undistorted endoplasmic reticulum. Small secretory vacuoles were occasionally seen, but virus was not detectable i n t r a or extra c e l l u l a r l y (Fig. 13). Fig. Ik. V i r u s - l i k e p a r t i c l e s at the c e l l membrane of chick embryo fib r o b l a s t s at the time of inoculation. Magnification xl05,000. Samples taken at the time of inoculation had s t r u c t u r a l details i d e n t i c a l to the uninfected control. One section contained a clump of v i r u s - l i k e p a r t i c l e s attached to an invagination i n the c e l l membrane -- apparently i n the process of being phagocytozed (Fig. lh). Evidence of virus r e p l i c a t i o n was f i r s t observed four hours after inoculation. At t h i s time virus precursor-like p a r t i c l e s of hi nm mean diameter were seen associated with i n t r a c e l l u l a r membranes, vacuoles, and r a r e l y with the c e l l membrane. These were smaller than BUN virus and larger than ribosomes (Fig. 15). At 12 hours mature v i r u s - l i k e p a r t i c l e s of mean external diameter 8h nm could be found e x t r a c e l l u l a r l y . These contained electron dense nucleoids of hh nm diameter. These mature p a r t i c l e s seemed to develop by budding into vacuoles and by budding from the c e l l surface.. Figure l 6 shows a bud with a dense core surrounded by a diffuse cyto-plasmic coat. Free virus was not observed i n the cytoplasm and no evidence was found of nuclear involvement. Wide-spread c e l l u l a r destruction with corresponding high levels of ext r a c e l l u l a r p a r t i c l e s was evident on the second day (Fig. 17). C e l l boundaries became discontinuous, empty vacuoles were numerous, a large amount of c e l l u l a r debris was free i n the media, and nuclear and mitochondrial degeneration was prominent. ..A. small number of vacuoles contained single or multiple v i r u s - l i k e structures (Fig. 18). Ext r a c e l l u l a r virus had an external diameter of 100 nm and dense i n t e r n a l core of k8 nm mean diameter. Fig. 15. Precursor-like p a r t i c l e s associated with i n t r a c e l l u l a r membranes i n chick embryo fib r o b l a s t s four hours post infection. Magnification x62,580. Fig. l 6 . Budding p a r t i c l e at the surface of a chick embryo fi b r o b l a s t c e l l 12 hours after infection. Magni-f i c a t i o n xl!5,000. c) F e r r i t i n Labelled H.Ep. 2 Cells Uninfected control H.Ep. 2 c e l l s showed no sign of virus or . cytopathic effect by electron microscopy. When control c e l l s were labelled, with a f e r r i t i n - a n t i BUN serum conjugate there was ..a minimal random non-specific staining of 'cellular and ex t r a c e l l u l a r material. The l a b e l l i n g procedure caused d i s t o r t i o n and destruction of c e l l s and u l t r a s t r u c t u r a l components, but c e l l boundaries, nuclei and mito-chondria were recognisable. Infected c e l l s contained v i r u s - l i k e p a r t i c l e s i n vacuoles and e x t r a c e l l u l a r virus by one day post infection. F e r r i t i n l a b e l l i n g of i n t r a c e l l u l a r virus components was not successful. C e l l components such as ribosomes re a d i l y took up the heavy metal st a i n and interfered with v i s u a l i z a t i o n of the smaller, less electron-dense f e r r i t i n p a r t i c l e This l i m i t e d the technique's use to the l a b e l l i n g and i d e n t i f i c a t i o n of ext r a c e l l u l a r BUN virus. Section from the one day sample showed isolated virus p a r t i c l e l a b e l l e d with antibody (Fig. 19). F e r r i t i n tagging of i n t r a c e l l u l a r virus could not be demonstrated. d) Mosquito Tissue Despite r e l a t i v e l y high virus levels as demonstrated by i n f e c t i v i t y t i t r a t i o n s , u l t r a s t r u c t u r a l evidence of virus m u l t i p l i c a -t i o n was very rare. Attempting to accomplish t h i s on the basis of cytopathic effects i s not sound as uninfected insect tissue contains 1+8 Fig. 17. Extr a c e l l u l a r p a r t i c l e s i n chick embryo f i b r o b l a s t cultures two days after inoculation with Bunyamwera virus. Magnification x79?200. Fi g . 18. Virus containing vacuole i n a Fig. 19. F e r r i t i n l a b e l l e d extra-chick embryo f i b r o b l a s t two days c e l l u l a r virus i n a after inoculation. Magnification H.Ep. 2 culture 2k hours x57,750. after infection. Magni-f i c a t i o n xl05,800. Fig. 20. Midgut c e l l s from uninfected Aedes aegypti. Magnification x20,000. mf: myelin f i g u r e - l i k e structure structures similar to those found i n arbovirus infected mammalian c e l l s , f o r example, the myelin f i g u r e - l i k e material within the gut c e l l s i n Figure 20. V i r u s - l i k e p a r t i c l e s with core diameters of 4-5 nm were seen e x t r a c e l l u l a r l y i n f a t body tissue surrounding the s a l i v a r y glands of mosquitoes which had been infected f o r 12 days. Figure 21 shows a c e l l from t h i s area with a large vacuole containing membranous struc-tures and v i r u s - l i k e material. Salivary glands and gut tissue from a 12 day mosquito showed 80 to 100 nm p a r t i c l e s within the cytoplasm and l i n i n g vacuolar membranes, but f a u l t y staining obscured s t r u c t u r a l d e t a i l and made i d e n t i f i c a t i o n d i f f i c u l t . Figure 22 shows a large vacuole i n tissue associated with the gut muscle of a mosquito which had been infected f o r 10 days. In addition to non-specific membrane structures i t contains p a r t i c l e s with 42 nm cores surrounded by d i s -torted envelopes of 83 nm to 125 nm diameter. Fig. 22. V i r u s - l i k e p a r t i c l e s i n mosquito gut 10 days after virus ingestion. Magnification x50,kOO. Discussion Discussion These results show that the prototype s t r a i n of Bunyamwera virus conforms to the accepted d e f i n i t i o n of an arbovirus by multiply-ing i n vertebrate- and insect tissue and by being transmitted from host to host by a mosquito. Ogunbi has demonstrated similar properties using the Ukawa s t r a i n of Bunyamwera. (53) The insect-mediated mechanical transmission of virus seen with some plant diseases does not play a role i n arbovirus infections. M u l t i p l i c a t i o n of virus within mosquitoes res u l t i n g i n high t i t r e s within s a l i v a r y tissue and secretions i s necessary f o r transmission. (17) The concentration of virus required to infect a mosquito varies greatly from species to species and i f t h i s . i n f e c t i o n threshold i s high enough i t e f f e c t i v e l y prevents a mosquito from being an arbovirus . vector. {lk, l6) The threshold phenomenon seems to be dependent on virus s u s c e p t i b i l i t y of 'gut c e l l s as m u l t i p l i c a t i o n must take place here p r i o r to hemolymph dissemination to non-central tissues, (kk) Arboviruses may be transmitted by mosquitoes other than t h e i r natural vectors and certain Worth American mosquitoes have shown the capacity to transmit viruses not endemic i n Worth America. (59?62) A l l Aedes species used i n t h i s research were e a s i l y infected by feeding on mice with a r t i f i c i a l yiremias. Since i n f e c t i o n threshold levels were not determined and blood virus levels were one hundred f o l d higher than those found i n mice with overt Bunyamwera encephalitis t h i s should be interpreted cautiously with regard to natural host-vector systems. The i n a b i l i t y of virus fed Aedes canadensis and vexans to transmit Bunyamwera to highly susceptible hosts (suckling mice) tends to rule out t h e i r p o t e n t i a l involvement i n natural Bunyamwera cycles. Trans-mission of Bunyamwera group viruses by B r i t i s h Columbian mosquitoes could prepare an ecological niche suitable for North American Tensaw and Cache Valley viruses. Intrathoracic i n j e c t i o n of virus by-passes the gut barr i e r and allows mosquitoes to support the growth of agents which are not i n f e c t i v e by ingestion. Even tissues of non-hematophagous insects such as grasshoppers, houseflies and carpet beetles can support arbo-virus growth after i n j e c t i o n , as can tissue culture c e l l s derived, from the moth Antheraea eucalypti. (35,72) Following the i n j e c t i o n of virus into the thoraees of Aedes  canadensis and vexans there was a decline i n .titratable v i r u s . This correlates with previous findings and may be due to a combination of virus i n a c t i v a t i o n and,eclipse phase. (lh,k3,hh) After ten days incu-bation, pools of whole mosquitoes showed virus levels approximately one hundred f o l d greater than the t i t r e of the inoculum. Salivary tissue t i t r e s increased one thousand f o l d and gut t i t r e s increased one hundred f o l d over a ten day period. Virus r e p l i c a t i o n rates and peak t i t r e s were highest i n the s a l i v a r y glands. Aedes vexans and canadensis were capable of maintaining the h 10 mouse LD„ l e v e l of virus ingested f o r eight days. This i s in d i c a -50 t i v e of a low l e v e l of m u l t i p l i c a t i o n without which a l l i n f e c t i v i t y would be l o s t due to virus inactivation. (lh) Bunyamwera levels i n these w i l d mosquito pools dropped over tenfold by day 17. Danielova noted a similar decrease with Tahyna i n Aedes vexans, which was probably due to mosquito aging or a form of immunity rather than virus induced pathology. (26) After two days Aedes aegypti injected with Bunyamwera showed virus t i t r e s three hundred f o l d higher than Aedes vexans and canadensis which received a similar virus dose. Peak t i t r e s were comparable, but were attained s i x days e a r l i e r i n Aedes aegypti. This correlates w e l l with the differences i n Ukauwa virus production between Worth American w i l d mosquitoes and Aedes aegypti. (53) Virus was constantly present i n the gut of Aedes aegypti following an i n f e c t i v e meal. After a one day lag corresponding to the time required for. gut m u l t i p l i c a t i o n and subsequent virus release into the hemolymph, i n f e c t i v i t y was detected i n other regions. The lag i n virus dissemination was very short compared to that found i n other mosquito-virus systems. (26,53) This may be due to an exceptional s u s c e p t i b i l i t y of Aedes aegypti gut to the prototype Bunyamwera st r a i n , (Fig. 11 & Fig. 12) and/or the high virus t i t r e s ingested from a r t i f i -c i a l l y viremic mice. (44,75) The detection of virus i n hemolymph samples and the i n i t i a t i o n of rapid r e p l i c a t i o n i n the s a l i v a r y glands occurred at the same time. Transmission was not accomplished at four days post i n f e c t i o n but a tenfold higher s a l i v a r y t i t r e at ten days resulted i n transmission to suckling mice. Chamberlain and Sudia showed that pre-vention of virus dissemination to sa l i v a r y tissues greatly reduced the transmission rate and high virus levels i n s a l i v a r y secretions are necessary f o r transmission. (17) In Uganda, Bunyamwera was transmitted by Aedes circumluteolus but evidence presented here suggests that Aedes aegypti would make an excellent vector i n urban areas. (31,37, 38,66) The adaption to mice of Chernesky's intravenous virus i n j e c -t i o n a r t i f i c i a l viremia. technique produced an instantaneous viremia one hundred f o l d greater than that, attained by mice with Bunyamwera encephalitis. Within one ha l f hour over 75$ of mosquitoes exposed to these mice had taken blood meals versus hOfo for a three-hour period when blood-virus soaked sugar cubes were used. Although the blood virus clearance rate of Bunyamwera i n mice i s approximately twice the rate of thermal i n a c t i v a t i o n at room temperature, the s i x f o l d longer time used for sugar-cube feedings results i n a less controllable v a r i a t i o n i n meal i n f e c t i v i t y from mosquito to mosquito. With lengthy methods the p o t e n t i a l f o r ingestion of virus below the i n f e c t i o n thres-hold i s also increased giving r i s e to p o t e n t i a l error i n virus develop-ment studies. Because the c i r c u l a t o r y volume'of mice can be calculated i t i s possible to produce a controlled l e v e l of viremia using a virus suspension of known i n f e c t i v i t y . Such a system could be as useful i n the accurate determination of mosquito in f e c t i o n thresholds as i t has been for Powassan virus i n Dermacentor andersoni t i c k s attached to rabbits. (23) Infected vertebrate tissue cultures incubated at 29°C gave comparatively rapid growth cycles with c e l l destruction and subsequent loss of detectable virus after four days. An i n i t i a l drop i n virus t i t r e corresponding to that seen i n mosquitoes was followed by a phase of rapid p r o l i f e r a t i o n concurrent with the appearance of cytopathic effect. Cytopathic effect was not found i n mosquito tissue even when comparable levels of virus were being produced. Chamberlain and Sudia have shown that the rate of virus production i n mosquitoes i s subject to thermal regulation and that a temperature of 29°C i s optimal for virus growth with mosquito longe-v i t y . (15) Bunyamwera produced similar growth curves i n 29°C chick embryo fib r o b l a s t s (Fig. 7) and mosquito tissue (Fig. 8, 9510,11,12) as opposed to the findings i n 37°C H.Ep. 2 and chick embryo fi b r o b l a s t s . Cytopathic effect i n the 29°C c e l l s was f i r s t seen four days after the onset of virus production. This suggests that to a degree, tempera-ture effects are responsible for the slow production of virus and lack of cytopathic effect seen i n most insect tissues. Bunyamwera replicated faster and produced higher t i t r e s i n mosquito organ cultures than i n whole mosquitoes. This suggests the presence of a growth l i m i t i n g factor i n whole mosquitoes. Electron microscopy of infected chick embryo fi b r o b l a s t s demonstrated a reproduction cycle similar to Murphy's findings i n Bunyamwera infected mouse brain. (51,52) Virus entry appears to be by phagocytosis. Cytopathology seen at four hours involves virus precursor-like p a r t i c l e s i n conjunction with i n t r a c e l l u l a r membranes not described by Murphy. The observation of complete virus at 12 hours correlates w e l l with the onset of. i n f e c t i v e virus production (Fig. 6). Maturation occurs by budding into cisternae or from the c e l l surface i n a manner similar to group A arboviruses. (2,50) Virus r e p l i c a t i o n leads to c e l l vacuolation and cisternae containing virus may be seen near the c e l l ' s surface p r i o r to releasing t h e i r contents into the ex t r a c e l l u l a r space. Nuclear involvement and free nucleoids within the cytoplasm were not seen. The diameter of the virus envelope varied from 80 to over 100 nm i n diameter depending on variations i n electron microscopy pre-paration and the type of host tissue used. The dense nucleoids, however, remained r e l a t i v e l y constant at k-5 ± 3 nm diameter. Immunotracing with f e r r i t i n l a b e l l e d anti-Bunyamwera serum i d e n t i f i e d similar p a r t i c l e s i n infected H.Ep. 2 c e l l s as Bunyamwera viruses. The presence of arboviruses within insect tissues i s r a r e l y confirmed by electron microscopy, and only Semliki Forest virus infected mosquito s a l i v a r y glands demonstrate cytopathic effect by l i g h t micros-copy* C+9) Wh i t f i e l d and Murphy have shown that complete Eastern Equine Encephalomyelitis virus i s not observed i n mosquito sa l i v a r y glands u n t i l four days after; the end of the e x t r i n s i c incubation period. A further eight day period i s required before these p a r t i c l e s are numerous. (76) Cytoplasmic p a r t i c l e s the size of Bunyamwera virus were seen 5.0 i n s a l i v a r y tissue containing 10 * mouse of Bunyamwera. Tissue from the f a t body and gut associated c e l l s of infected mosquitoes demonstrated that virus accumulates i n vacuoles as i t does i n verte-brate tissue. No evidence was found of the nuclear involvement reported by Janzen. (36) The prevalence of virus i n the f a t body near the sal i v a r y glands suggests that, care should be taken to avoid contamination when sali v a r y glands are being dissected out f o r t i t r a t i o n . Literature Cited Bibliography 1. Abdelwahab, K. W. E., Almeida, J. D., Doane, F. W. and McLean, D. M. 196U. Powassan virus: morphology and cytopathology. Canad. Med. Ass. J. 90: 1068-1072. 2. Acheson, N. H., Tamm, I. I967. Replication of Semliki Forest virus: an electron microscopic study. Virology 32: 128-1*4-3. 3. The American Committee on Arthropod-borne Viruses. 1969. Arbovirus names. Amer. J. Trop. Med. Hyg. 18: 731-73*+. *+. Anderson, S. G. and Ada, G. L. 1959- Some aspects of the reac-t i o n between crude Murray Valley encephalitis (MVE) virus and deoxycholate. Virology 8: 270-271. 5. Anderson, ¥. A. D. 1966. Pathology, Vol. 1. pp 278-280. C. V. Mosby Co., Saint Louis. 6. Bates, M. and Roca-Garcia, M. 19*4-6. The development of the virus of yellow fever i n Haemogogus mosquitoes. Amer. J. Trop. Med. 2 6 : 585-605. 7. Bergold, G. H. and Weibel, J. 1962. Demonstration of yellow fever virus with the electron microscope. Virology 17: 55*+-562. 8. Black, L. M. 1950. A plant virus that m u l t i p l i e s i n i t s insect. vector. Nature l 6 6 : 852-853. 9. Brown, J. G. and Jones, R. H. I966. Observations on blue tongue virus i n the s a l i v a r y glands of an insect.vector, Culicoides  variipennis. V i r o l o g y 30: 127-133. 10. Casals, J. 1957- Viruses: the v e r s a t i l e parasites: the arthropod-borne group of animal viruses. Trans. N. Y. Acad. Sci. 19: 219-235. 11. Casals. J. and Whitman, L. i 9 6 0 . A new antigenic group of arthropod-borne viruses. The Bunyamwera group. Amer. J. Trop. Med. and Hyg. 9: 73-77. 12. Casals, J. Personal communication. 13. Causey, 0. E., Causey, C. E., Maroga, 0. M. and Macedo, D. G. 1961. 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