The utinga virus: antigenic characterization and serologic epidemiology

Transcription

1 Yale University EliSchlar A Digital latfrm fr Schlarly ublishing at Yale Yale Medicine Thesis Digital Library Schl f Medicine 1967 The utinga virus: antigenic characterizatin and serlgic epidemilgy Ihr G. Zachary Yale University Fllw this and additinal wrks at: art f the Medicine and Health Sciences Cmmns Recmmended Citatin Zachary, Ihr G., "The utinga virus: antigenic characterizatin and serlgic epidemilgy" (1967). Yale Medicine Thesis Digital Library This pen Access Thesis is brught t yu fr free and pen access by the Schl f Medicine at EliSchlar A Digital latfrm fr Schlarly ublishing at Yale. It has been accepted fr inclusin in Yale Medicine Thesis Digital Library by an authrized administratr f EliSchlar A Digital latfrm fr Schlarly ublishing at Yale. Fr mre infrmatin, please cntact elischlar@yale.edu.

2

3 YALE MEDICAL LIBRARY

4

5

6 THE UTING-A VIRUS: Antigenic Characterizatin and Serlgic Epidemilgy B.S. Ihr G. Zachary Trinity Cllege 1963 A Thesis resented t the Faculty f the Yale University Schl f Medicine in artial Fulfillment f the Requirement fr the Degree Dctr f Medicine The Department f Epidemilgy and ublic Health April 1967

7 &?sg?

8 ACKNWLEDGMENTS I wish t express my deepest gratitude t Dr. Rbert E. Shpe fr his cntinued encuragement, advice, critical appraisal, and guidance f this prject. I als wish t thank Yale University, The Rckefeller Fundatin and Dr. Wilbur G. Dwns, Directr f The Yale Arbvirus Research Unit, fr making it pssible fr me t receive the Research Fellwship under which the study in Brazil tk place. I am indebted t Dr. Jhn. Wdall, Directr f the Belem Virus Labratry, fr making available t me the facilities f that labratry, and t Miss Amelia Andrade fr valuable instructin and assistance.

9 Digitized by the Internet Archive in 217 with funding frm The Natinal Endwment fr the Humanities and the Arcadia Fund

10 CNTENTS 1 INTRDUCTIN MATERIALS AND METHDS VIRUSES. 6 HEMAGGLUTINATIN-INHIBITIN TESTS. 6 NEUTRALIZATIN TESTS. 13 CMLEMENT-FIXATIN TESTS. 17 RESULTS CRSS-HI TESTS. CRSS-CF TESTS. CRSS-NT TESTS. RESULTS F SERLGIC SURVEY IN BELEM. NEUTRALIZATIN TESTS DISCUSSIN. 26 SUMMARY TABLES AND FIGURES REFERENCES

11 * ** * fcial*** *>«CA«I3 -I '-fffntj M -». C <i» e a <» «!» ; f» * -1 5 «% 5 u >!*«! r> * ' '> < - B

12 INTRDUCTIN The Utinp:a Virus In ctber f 1965 a virus was islated frm the bld f the three-ted slth (Bradypus tridactilus) captured in the Utinga Frest near Belem. Serlgical testing f this virus (BE An 84735) against grup specific immune sera f all presently knwn viruses f that regin shwed that this agent was serlgically related t the Simbu grup f arbviruses, but was nt identical t any f the knwn members f this grup. 42 The serlgical relatinship f Utinga virus t rpuche virus f the Simbu grup was especially interesting, since rpuche had been assciated with a large epidemic in Belem during the Spring f rpuche virus was islated frm 14 febrile cases, and f the patients studied, 42 f 87 cnverted frm negative t psitive during the epidemic with respect t neutralizing antibdies. c The rpuche virus had been islated frm man fr the first time in Trinidad, a few years prir t the epidemic in Belem.2> When the newly islated agent frm the Utinga Frest (subsequently named the Utinga virus) was nted t be related t rpuche virus, it became interesting t speculate whether it culd have played any rle in the rpuche epidemic.

13 I '

14 2- - The study as presented in this paper was undertaken with three main bjectives in mind: (1) t characterize this new agent, the Utinga virus, with respect t its serlgical reactivity and its relatinship t the ther members f the Simbu grup f Arbviruses; (2) t btain sme idea f the serlgic epidemilgy f this virus and its prevalence; and (3) t find ut its pssible asscia tin with the rpuche virus, whether humans infected with rpuche during an epidemic als develp Utinga antibdy, and whether it may have played any rle in human illnesses in the Belem area ver the last few years. The Arbviruses - G-eneral Cnsideratin The arbviruses, Jf arthrpd-brne animal viruses, are a large hetergeneus grup f viral agents linked tgether in classificatin by their capacity t infect certain vertebrates - mammals, reptiles and birds - and t multiply in the bdy f arthrpds withut prducing, with rare exceptins, any pathlgical changes in these infected vectrs. Such agents are maintained in nature by a cntinuus cycle in which the arthrpd vectr becmes infected, generally by ingesting bld frm a vertebrate hst at a time when the virus circulated in the latter, and, after a perid f days, designated as the extrinsic incubatin, the vectr, by biting can transmit the disease t a new susceptible hst * fficially accepted nmenclature by the subcmmittee n virus nmenclature at the Internatinal Cngress f Micrbilgy, Mntreal, August 1962.

15

16 3- - Besides the cmmn bilgical cycle, certain ther characteristics are prbably cmmn t mst f these agents: (l) an essential lipid envelpe making the particle rather highly sensitive t ether and sdium desxychlate, and (2) an RNA cre. Als, they are in general rather unstable viruses, readily inactivated by relatively little heat (37-57 C), and all prduce encephalitis in suckling mice fllwing intracerebral inculatin. 5,19 T the present time abut 22 different arbviruses have been islated in varius parts f the wrld and the list is expanding yearly. Thrugh the use f serlgical tests - neutralizatin (NT), cmplement-fixatin (CF), and hemagglutinatin-inhibitin (HI)- mst f these viruses have nw been classified int 22 antigenic grups, principally by Casals and c-wrkers. ^ ^9 <f Sme f these grups als cntain subgrups f viruses shwing very clse relatinship with each ther. Althugh many labratries and wrkers arund the wrld have cntributed t the ever grwing list f arbvirus islates, and t the elucidatin f the epidemilgy f these agents, there are several labratries which have been exceptinally prductive in this respect. ne f these labratries is the Belem Virus Labratry (Belem, Brazil) a field labratry maintained by the Bckefeller Fundatin and the Brazilian gvernment since 1954, which has been respnsible fr the islatin f at least 35 new IQ agents and many already knwn agents. 1 * 1 A zrx The

17

18 4- - functining f this labratry is explained, in detail by i2 Causey et al. ver the years this labratry has accumulated a vast number f sera (human, mammal, bird and reptile) which prvide a wealth f material fr retr spective serlgical studies. Belem - The Study Area Belem and the surrunding area invlved in the study, as shwn in Figure 1, are situated n the right bank f the ara River, extending nrth frm the G-uama River t the sea, and abut 5 inn inland. In this area the climate is trpical rain frest, and is characterized by relatively high levels f temperature and rainfall, with minimal annual variatin. The mean mnthly temperatures varies less than 5 C during the year, and the driest mnth has at least 6. cm. f rainfall. 12 East f Belem, between 5 and 2 km., is a cntinuus tract f frest alng the G-uama River. This is divided int the ribca virgin frest with many swamps and streams, the prperty f the Institut Agrnmic d Nrte (I.A.N.), the site f an ld cca plantatin, nw reverted t dense secnd grwth, and the Utinga frest, a watershed fr the city reservirs with several large, deep lakes. The Simbu G-rur? At the present time 11 different viruses are knwn in the Simbu grup f arbviruses, and mst f these have been

19 t *. >.

20 5- - islated nly within the last 1 years in varius parts f the wrld. Althugh antibdies in humans have been detected fr Manzanilla, Simbu, and rpuche viruses, f all the Simbu grup viruses nly the rpuche has been shwn t cause any human disease, and this agent is als the nly ne, ther than the Utinga virus, that has been islated in the Belem area. The ther members f this grup, with the dates and lcatin f islatin, and the species frm which islated are listed in Table I.

21 -Cr Jt

22 6- - MATERIALS AND METHDS VIRUSES The fllwing Simbu grup viruses were used in testing: Strain Virus assap;e level Akabane Ja Ar Buttnwillw A Ingwavuma SA An Manzanilla TRVL rpuche TRVL Sathuperi IG Simbu SA Ar Utinga BE An Sang lb An lb An Yaba 7-5 HEMAGGLUTINATIN-INHIBITIN TESTS The majr part f the serlgical survey was dne emplying the hemagglutinatin-inhibitin (HI) test. technique used was that described by Clarke and Casals The 1 5 with sme mdificatins. reparatin f Hemagglutinating Antigens The HA antigens were prepared frm the brains f 2-4 day ld mice inculated by the intracerebral rute with.2 ml. f a 1 muse brains. _2 dilutin f stck infectius suckling- The mice were harvested (n the eighth day in the case f Utinga virus) when sme f the mice began t die, and many thers appeared sick frm the infectin. The har vested mice were kept in plyethylene bags at -7 C until ready fr use. The HA antigen was prepared by the sucrse-acetne 1 5 extractin methd. This prcedure has been used increasingly

23 . ) *

24 ver the last few years at the Rckefeller Labratries and is prving t be superir t all ther methds. The remved and weighed suckling-muse brains were hmgenized with fur vlumes f a chilled 8.5$ aqueus slutin f sucrse, and the hmgenate added, by squirting frm a syringe, t twenty vlumes f chilled acetne. This mixture was shaken vigrusly, and the supernatant decanted. The same vlume f chilled acetne was added t each bttle, and the preparatin was allwed t stand in an ice bath fr ne hur. The supernatant was then discarded and the sediment dried in a vacuum apparatus. The dry pwder was rehydrated with.85$ saline, the vlume used being equal t.4 f the ttal vlume f hmgenate used, and the pre paratin was left vernight t rehydrate. It was then centrifuged fr 3 minutes at 1, rpm. The supernatant was distributed t ampules (.5 ml), lyphilized, and the ampules sealed and kept at 4 C until ready fr use. Abut 9 ampules f Utinga antigen were transprted, withut refrigeratin during a perid f abut tw weeks, t Brazil. Hemagglutinatin testing upn arrival in Brazil demnstrated that in spite f the prlnged expsure f the antigen t ambient temperatures, there was n lss f titer. In Brazil, the antigen was kept at 4 G withut lss f titer ver 3 mnths. Sera and Ascitic Fluids Mst f the mammalian sera examined in the survey were btained frm the mammals captured in the Utinga frest,

25 ,, '

26 8- - and mst f the bird sera frm birds frm the I.A.N. The human sera frm area. were cllected frm inhabitants f several districts f Belem during a serlgical survey fr antibdies t the rpuche virus. The human sera frm all subsequent years were cllected frm cases f fevers f unknwn rigin (FU) sented themselves t the labratry; which pre these were in many cases emplyees f the labratry and their families. Sme f these cases were frest wrkers, many f whm als lived in the frest. All the sera tested had been stred at the Belem Virus Labratry fr varius lengths f time several years) (a few days t at -2 C r -7 C. Reference immune sera and muse ascitic fluids were made by a series f 3 t 5 weekly intraperitneal inculatins in adult mice f.1 ml f infected suckling- muse-brain suspensin prepared in saline and Freund's cmplete adjuvant (Difc Labratries). After several i.p. inculatins with the antigen, mice were inculated with.1 ml f ascitic fluid frm mice with ascites secndary t Sarcma 18/TG-. mice develped ascites the (1-14 days) When these the ascitic fluid was remved using a 5 cc syringe with an 18 G needle. Mice that survived this prcedure, were paracentesed again at intervals f a few days. The ascitic fluid was centri fuged fr 1 minutes at 1, rpm t remve tissue debris, and the supernatant fluid was used as reference immune ascitic fluid in all subsequent HI, GF, and NT tests.

27 . i,

28 9- - The Sarcma 1 8/TG- is a slw-grwing strain f the tumr which was riginally selected fr resistance t the synergistic antineplastic drug cmbinatin azaserine and 6-chlrpurine. 27 Since it develps mre slwly in mice, and.'.is much less invasive than the riginal Sarcma 18, as used by Herrmann and Engle and Tikasingh et al, cnsiderably larger vlumes f ascitic fluid can be btained. The immune ascitic fluids thus prduced have been shwn t have antibdy titers which cmpare very favrably with thse f the sera frm the same mice, t be f high specificity. and 27 reparatin f Sera fr HI Test All sera were treated by the acetne extractin methd 15 f Clarke and Casals ' t remve the nnspecific inhibitrs f arbvirus hemagglutinins, which, rterfield and Rwe 24 as bserved by, many sera cntain. Rutinely.5 ml f serum was treated fr all HI tests.. % The serum was diluted 1:1 in 85 saline. Using a drpper pipette with a #23 dispsable needle the serum was squirted int 12 vlumes chilled acbtne; the prepara tin was shaken and allwed t stand in an ice bath fr five minutes. at 1, rpm, The tubes were then centrifuged ne minute the supernatant decanted, and the precipitate resuspended by shaking. An additinal 12 vlumes f acetne was added and the preparatin was allwed t stand fr ne hur in an ice bath. The tubes were then

29 ' «* *

30 1- - centrifuged 5-8 minutes at 1,5 rpm, the supernatant decanted, and the precipitate in the tubes was dried fr ne hur in a vacuum jar at rm temperature. pwder was rehydrated with.5 ml. (1 vlumes with respect t the riginal vlume f serum) ph 9. slutin. The dry f brate-saline This gave a final serum dilutin f 1:1 based n the vlume f serum initially intrduced. Since it has been shwn by rterfield 23 that many sera cntain naturally ccurring agglutinins fr gse erythrcytes, all sera were rutinely absrbed with such cells befre being used in the tests. The gse erythrcytes were btained by bleeding geese kept at the labratry, with the bld cllected in 5 cc syringes with acid-citrate-dextrse (AGD). fr 1 minutes at 1, rpm, The bld was then centrifuged the plasma aspirated ff by a 1 cc pipette, and the cells then washed three times with saline, each time fllwed by centrifugatin fr 1 minutes at 1,5 rpm. The final vlume f packed cells was diluted with 5 vlumes f.4$ Bvine-albumin in brate-saline (final dilutin 1:6). T each serum (.5 ml) was added.6 ml f the 1:6 dilutin f gse erythrcytes. This preparatin was allwed t stand fr 2 minutes in an ice bath with ccasinal shaking. The tubes were then centrifuged fr 15 minutes at 1,5 rpm, used fr the HI tests, a 1:2 dilutin. the supernatant decanted, and the serum at this pint being at

31

32 The packed washed gse erythrcytes were als used fr preparing the cell suspensin used in the HI test prper. The packed cells were diluted 1:8 with dextrse- gelatin- vernal (DGV) suspensin f cells. and kept as such as a stck By testing the iaemagglutinating antigen f the Utinga virus at varius phfs frm 6. t 7. it was fund that the antigen shwed ptimum agglutina tin at ph f apprximately 6.4jand therefre this ph was used in all subsequent HA and HI tests. adjusting-diluent (VAD) The virus slutin f the desired ph (r apprximately 6.4) was prepared by cmbining phsphate buffer slutins f ph 6. and ph 7. in the prprtins f 6:4. G-se cells were added t the VAD slutin in the prprtin f ne part cells (at 1:8) per 39 parts diluent at the desired ph. This final preparatin was used fr the HA and HI tests. Methds fr the HA and HI Tests All the HA and HI tests were carried ut using the micrtechnic which is based n the mdificatin by Sever 28 f a micrtitratin methd riginally intrduced by Takatsy. This micr methd has the advantage f facility f perfrmance and cnservatin f reagents and sera. It emplys small plastic plates with rws f wells (see Figure 2) in which the reactins are carried ut, wire lps fr making the dilutins. and calibrated

33 . 1.

34 reliminary tests using bth, the lder macr methd f tube dilutins, and the micr methd with lp dilutins 23 2Q cnfirmed the findings by Sever 1 and Shpe J that these methds have cmparable sensitivities and accuracy. All the sera were tested in 1 r 2 dilutins against ne dilutin f antigen adjusted t give abut 4 units f hemagglutinin per.1 ml. The Utinga virus antigen used gave 4 units f hemagglutinin at a dilutin f 1:4 and this was the dilutin used fr mst f the tests. The number f units f hemagglutinin is calculated by cnsidering the highest dilutin f antigen which gives cmplete r nearly cmplete agglutinatin as giving ne unit f hemagglutinin per given vlume. All dilutins f sera and antigen were made in bvinealbumin-brate- saline ph 9. slutin. All cmpnents f the test were distributed n the plates using a drpper pipette with a blunt 18 gauge needle calibrated t give.25 ml per drp. The acetne treated sera at 1:2 dilutin were distributed in the wells, and ne 24-fld dilutin made with the wire lps (1:4 dilutin). ne cntrl well was used fr each serum t rule ut any nn-specific gse cell agglutinins in the sera. Diluted antigen, giving 4 units f hemagglutinin per.25 ml was then distributed t all the wells except the cntrls. With each test a titratin f the antigen was dne starting with the dilutin f the antigen as used fr the

35 * '

36 -13 test. A cntrl f the diluent was als dne with each test, t exclude the pssibility f nn-specific agglutinatin by the diluent. The plates were then incubated vernight at 4 C. The next day the gse erythrcyte suspensins were prepared at ph 6.4 in VAD slutin, 2 drps (.5 ml) n the plates. as explained abve, and f the cells were added t all the wells The plates were gently shaken by tapping, incubated at 37 G fr 45 minutes and read. The plates were read as + = cmplete hemagglutinatin (n hemagglutinatininhibitin); + = partial hemagglutinatin hemagglutinatin-inhibitin); (trace ± = trace hemagglutinatin (partial hemagglutinatin-inhibitin); = n hemagglutinatin (cmplete hemagglutinatin-inhibitin). nly sera shwing cmplete inhibitin were cnsidered psitive. All sera giving cmplete r partial hemagglutin atin-inhibitin were retested using six serial 2-fld dilutins f sera: 1:2-1:64. NEUTRALIZATIN TESTS Neutralizatin tests in micee were carried ut t substantiate the findings f the HI tests. Grss- neutralizatin tests in mice were perfrmed t further elucidate the relatinships f the Utinga virus t the ther members f the Simbu grup. The neutralizatin test is based n the principle that when a specific immune serum is added t its crrespnding virus, the virus is rendered nninfective r is "neutralized.

37 .

38 14- - While varius animals are used fr neutralizatin tests, the Swiss muse has been fund t be a suitable animal fr wrk with arbviruses. This animal has the advantage bth frm the standpint f cst, since fr these tests large numbers f animals have t be used, and frm the stand pint f hst susceptibility, since it has been shwn that all currently knwn arbviruses prduce infectin and disease in suckling mice when inculated by the intracerebral (IG) rute.d y This susceptibility was first demnstrated with yellw fever virus by Theiler in The methds used fr the present study were thse as emplyed currently at the Yale Arbvirus Research Unit and are utlined briefly belw. The albin Swiss mice, Charles River strain, were used exclusively fr all tests. Since sme agents are pathgenic nly fr the very immature muse, even by the intracerebral rute, and since the Utinga virus appears t have a fairly lng incubatin time (abut 6-1 days), nly 2-4 day ld mice were used. All the baby mice used fr any ne test were pled tgether in ne bx, carefully mixed, and distributed back randmly t the individual cages, eight mice per cage. The cnstant serum-varying virus methd was emplyed fr all tests. This methd has the advantage that it has been mre frequently used in viv, mre is knwn abut its variables, the end pints f titratin are rather well defined, and it is readily subject t standardizatin

39 . '

40 15- - fr reference t any cnstant r index, such as the neutralizatin index. The viruses emplyed were 1$ infectius sucklingmice brain in.75$ Bvine-albumin-phsphate ph 7.2 (BA). Tw types f virus preparatins were used. Fr sme f the tests a lyphilized preparatin f the abve was used which had been kept in sealed ampules at -2 C, and was rehydrated just prir t use with the apprpriate amunt (.5 r 1. ml) f.75$ Bvine-albumine-phsphate ph 7.2. Sme f the tests were dne using a 1$ brain in BA nt lyphilized, but kept in sealed ampules at -7 G. Just prir t use the 1$ infectius virus brain preparatin was rehydrated with.75$ BA, r simply thawed ut if the frzen material was used, and serial 1-fld dilutins made in tubes. All the muse immune ascitic fluids (prepared as described previusly) which were used fr the crss-neutral izatin tests were stred in lyphilized frm in sealed ampules at -2 C. The nly exceptins t this were the immune ascitic fluid t the Utinga virus and the nrmal ascitic fluid used fr cntrls. These were stred frzen withut lyphilizatin in plastic tubes at -2 C. The sera used in the neutralizatin tests dne in Belem, and the muse immune ascitic fluid t the Utinga virus used as cntrl in these tests, were stred frzen at -2 G. Just prir t use, the lyphilized ascitic fluids were rehydrated with.75$ BA, and the frzen ascitic

41 . '

42 -16- fluids r sera thawed ut at rm temperature. The ascitic fluids r sera were then distributed, by pipette t the apprpriate tubes. An equal vlume f the apprpriate virus dilutin was added frm the master tubes, and the tubes were incubated in a water bath fr 6 minutes at 37 C. During the time f inculatin the tubes were kept in an ice bath. N fresh animal serum (i.e., accessry factr) was used. Ascitic fluids were nt inactivated. Suckling mice (2r4 days ld) were inculated intracerebrally with.2 ml using.25 cc glass tuberculin syringes with 26 gauge needles. inculated with each dilutin. ne cage (8 mice) wa_s The mice were checked daily fr incidence f illness r death and dead mice were discarded. A daily recrd f mrbidity and mrtality was kept n standard "muse cards," (See Figure 3.) Calculatin f the Neutralizatin Index The results f the neutralizatin and crss neutralizatin tests were calculated n the basis f a 5 per cent end pint expressing a 5 per cent mrtality r LD^q (5 percent lethal dse). The methd f Reed and Muench2^ was used fr calculating the LD^q and the neutralizatin index. Althugh this methd is applicable primarily t a cmplete titratin series, that is, the whle reactin range, frm per cent t 1 per cent mrtality, it can be used even when these cnditins are nt fulfilled, if the reactins ccur in a unifrm manner ver the range f dilutins emplyed.

43 .,

44 -17- A hypthetical arrangement f data used in cmputing the LD5 titer by the Reed-Muench frmula is as fllws: Virus Dilutin Died Survived Died Accumulated Values Mrtality Survived Hati- er Gent /22 n/16 8/13 1 3/13 / The necessary prprtinate distance f the 5 per cent mrtality end. pint is btained as fllws: J.% mrtality at dilutin next abve 5% - (5/) {% mrtality at dilutin next abve 5% - {% mrtality at dilutin next belw 5%) = rprtinate Distance Fr the abve example the lg L5 = 3.5 The lg neutralizatin index, in which frm the results are presented, indicates the capacity f the serum t neutralize the actin f the virus n the hst system emplyed. It represents the difference between the titer f the virus in the presence f nrmal cntrl serum and the titer f the virus in the presence f the test serum, and is btained by subtracting the lgarithm f the LD^Q titer f the immune (r test) serum frm the lgarithm f the LDcjq titer f nrmal cntrl serum. GHLEMENT-FIXATIN TESTS The prcedure used fr the cmplement fixatin test was that emplyed presently at the Yale Arbvirus Research Unit (YARU) and is based n the methd described by Casals^4

45 '. it

46 with the later mdificatins by Fultn and Dumbell by the Rckefeller labratry. 1 and It is a micr methd emplying plastic plates with rws f small wells similar t the plates used fr the HI tests (see Figure 2), and thus has the advantage f cnservatin f redgents. Cmplement Titratin rir t each test the cmplement was titrated using the test tube methd (1x13 mra tubes), and again in the test itself n the micrplates, with the results alway in clse agreement by bth these methds. Vernal buffer, ph 7.4, was used fr making all the dilutins in the test and fr cntrls. In the F tests dne at YARU a cmmercial guinea pig cmplement was used (Baltimre Bilgical Labratry, Baltimre, Maryland), while in Brazil, frzen pled guinea pig serum was used as the surce f cmplement. Fresh cmplement at a dilutin f 1:3, was prepared just prir t each test and nine serial dilutins made in the fllwing manner: hi. 1 3* (1:3)ml. D 1 Vernal iiluent ml Sensitized sheep erythrcytes were prepared by cmbining equal vlumes f 4$ sheep erythrcytes (prvided as either a cmmercial 1$ sheep cell suspensin frm Baltimre Bilgical Labratry r as 1$ erythrcytes frm sheep kept at the labratry at Belem) with an equal

47 . : < *» '

48 -19- vlume f anti-sheep hemlysin at Bilgical Labratry, 1:8 (Baltimre Inc. West Chester, enn.). The hemlysin-cell suspensin was then incubated fr 15 minutes in a water bath at 37 C. The sensitized sheep cells were prepared in the same manner fr bth the cmplement titratin and fr the cmplement fixatin test..1 ml. f the sensitized sheep cells was added t nine tubes cntaining.2 ml f the serially diluted cmplement and.1 ml. f vernal diluent. The tubes were incubated fr 3 minutes in a 37 C water bath:, placed in the refrigeratr at 4 C fr abut ne hur t allw fr cmplete settling in the tubes, hemlysis) and read n a scale f (cmplete t 4 (n hemlysis). n the basis f the preliminary cmplement titratin the 1:3 cmplement slutin was adjusted t cntain 2 units f c* per.1 ml f 1:3 c*. In practice this adjustment was rarely fund necessary since the 1:3 c* as prepared, was usually f the exact cncentratin desired. Methd fr the CF Tests Fr the CF test each serum r ascitic fluid was diluted t 1:4 with vernal buffer and inactivated, at 6$C fr 2 minutes. Six serial 2-fld dilutins f each inactivated serum and f each antigen were made in test tubes starting at 1:4. The serum dilutins were distri buted n the plates (l pippttes with blunt 18 G- needles calibrated t give.25 ml. per drp. drp per well) using drpping ne drp f cmplement (1:3) was added t

49 ... - i.

50 each well, and then ne drp f the antigen dilutins t the apprpriate wells. With each test a serum cntrl, an antigen cntrl and a cmplement titratin were als included. The plates were incubated vernight at 4 C, and the fllwing mrning 1 drp f sensitized sheep erythrcytes was added t each well. The plates were gently shaken and incubated fr 3 minutes at 37-4 C with ccassinal shaking. At the end f this time the plates were placed in a refrigeratr t allw cmplete settling, and then read n a scale f hemlysis) t 4+ (n hemlysis). (cmplete

51 '

52 21- - RESULTS CRSS-HI TESTS The results f the crss-hi tests f the Utinga virus with the ther members f the Simbu grup are shwn in Table II. Cmplete crss-hi testing f all viruses against each ther was nt pssible because f the unavail ability f hemagglutinating antigens f all the viruses in the Simbu grup. viruses, As far as can be ascertained,sme f these including rpuche, Simbu and Yaba 7 d nt prduce hemagglutinating antigens. Sang, lb An 555, Akabane and Sathuperi d prduce HA antigens, but these were nt available at the time f testing. The results in Table Iliare expressed as the reciprcal f the highest dilutin f immune serum r ascitic fluid giving cmplete inhibitin f 4 r 3 units f hemagglutinin. The Utinga immune ascitic-fluid gave a titer f 1:32 and n ther immune ascitic fluid shwed any inhibitin f the Utinga hemagglutinating antigen. The Utinga, Manzanilla and Ingwavuma immune ascitic fluids shwed lw titer inhibitin f sme f the ther antigens. CRSS-CF TESTS The results f the crss cmplement-fixatin tests, dne in cnjunctin with Dr. R, E. Table III. Shpe, are shrn in Fr this testing an Utinga immune ascitic

53 .. X.

54 22- - fluid (BE An AF, Ii) prduced By nly ne intra- peritneal inculatin f virus with adjuvant, and an immune serum (BE An 84784, Ser.3i) prduced by three inculatins f virus with adjuvant were used. The results are expressed as the reciprcal f serum dilutin/reciprcal f antigen dilutin. ascitic fluid (AF) The Utinga shwed a lw-titer reactin in the hmlgus system and n crss reactin with any ther antigen. The Utinga hyper-immune serum which was anti cmplementary at 1:4 shwed a high-titer reactin in the hmlgus system (512/S 256) antigen (> 128/ 256), ther antigens. and with the rpuche and much less reactin with several n the ther hand the rpuche immune serum reacted at a high titer with its hmlgus antigen (128/64) but shwed a relatively lw titered reactin with the Utinga antigen (8/64). CRSS-NT TESTS Table IV shws the results f the crss-neutralizatin tests in mice with the Simbu grup viruses. The results are expressed as the lg neutralizatin index. In the hmlgus systems the Utinga immune AF gave the lwest lg neutralizatin index (1.6). The ther Simbu grup ascitic fluids neutralized 2.6 lg LD^q mre f their respective hmlgus viruses than the Utinga virus. Testing the Utinga ascitic fluid against all the viruses f the Simbu grup again shwed a lg neutralizatin

55

56 23- - index with the hmlgus virus f 1.6 which was ne lg r mre greater than the ther viruses. RESULTS F SERQLQG-IC SURVEY IN BELEM In all, 1336 sera were screened by HI testing fr antibdies t the Utinga virus. surces f the sera by genus, btained. frm Belem, f 885 sera, Table V, shws the species and dates when excluding the 1961 human sera nly 5 sera shwed psitive HI reactins at a 1:2 r greater dilutin, Table VI. Tw f these sera were f insufficient quantity fr any further testing,but the ther three were subjected t neutralizatin tests in mice against the Utinga virus (See Neutralizatin Tests). Fur hundred and fifty-ne human sera btained frm residents cf Belem during the rpuche epidemic in 1961, were tested by HI fr antibdies t the Utinga virus. Using the antigen which had been prepared at YARU, were fund giving hemagglutin inhibitin at greater dilutins. (psitive at 1:4) 18 sera 1:2 r f these 18, hwever, all except ne failed t inhibit the Utinga antigen which was being used at that time by the Belem labratry. This latter antigen had been prepared at the Belem labratry and had a 4-fld higher hemagglutinating titer, giving 4 units f hemagglutinin at a 1:16 dilutin (cf. YARU antigen 4 units f hemagglutinin at 1:4 dilutin). In these tests the hmlgus immune serum gave a titer f 1:8 with the YARU antigen, and 1:16 with the Belem antigen.

57 ^ J>:

58 24- - All the 18 sera were again prepared by the acetne extractin methd, and retested by HI with bth the YARU and Belem antigens. This time nly 6 f the 13 sera reacted at 1:2 with the YARU antigen and nne reacted with the Belem antigen. The hmlgus immune serum reacted at 1:4 and 1:8 respectively (cf. 1:8 and 1:16 in first test). Nne f these 18 sera reacted in CF with either rpuche r Utinga antigen. ne f the slth sera which had reacted at 1:4 in HI was als tested by CF, and did nt react with either the rpuche r Utinga antigens. These results are summarized in Table VII. NEUTRALIZATIN TESTS Neutralizatin tests in mice were perfrmed against the Utinga virus with sera frm the fllwing surces: (1) Sera btained in 1961 frm residents f Belem wh had experienced febrile illnesses during the rpuche epidemic and frm whse bld the virus was Islated. These sera represented the secnd r third bleedings, abut 1 r 2 mnths after the illness, and all neutralized and/r reacted in CF tests with rpuche. in HI with Utinga. 3 pigs) (2) Nne reacted Sera frm ungulate animals (1 bull, which in earlier testing by the Belem Labratry had shwn psitive HI reactins (at 1:2 r greater) Utinga antigen. with Tw f these animals had been brught in frm Maraj Island, acrss the bay frm Belem, Chaves and ne frm rt de edr, prximity t Belem. ne frm all areas in clse (3) Sera frm the 2 slths and 1

59 '

60 25- - marsupial which had reacted in HI tests with Utinga. (4) ne serum frm a labratry emplyee whse serum frm 1963 reacted in HI with Utinga. Since the 1963 sample was f insufficient quantity fr further testing, a sample btained 2 years later (1965) was used. This sample did nt react in HI. The results f the neutralizatin tests are shwn in Table VIII. The Utinga immune ascitic fluid gave a lg neutralizatin index (Belem 945) 2.4. f the 4 human sera nly ne shwed a suggestin f neutralizatin with a lg neutralizatin index f 1.6; the ther were all The tw slth sera and ne pig serum gave lg neutralizatin indeces f 1.2, 1.6 and 1.3 respectively and all the ther sera tested were less than 1..

61 ....

62 26- - DISCUSSIN When the Utinga virus was islated in 1965 the first step was t determine whether the islate was an arbvirus. Althugh the ideal and cnclusive evidence fr deciding that a virus belngs in this family wuld be the exper imental reprductin f the natural cycle, virus, hst, and vectr, invlving the this is seldm pssible. Utinga virus this wuld have been impssible, With the since as yet n vectr had been fund fr this agent. Amng the cnsideratins which cntributed t the inclusin f the Utinga virus with the arbvirus grup were the fllwing: 1. The virus was islated frm a lwer animal, captured in a trpical rain frest with abundant numbers f arthrpds. 2. It was cnsistently pathgenic fr baby mice. 3. It was inactivated by sdium desxychlate. 4. An agglutinin fr gse red bld cells was 43 demnstrated in infected muse brain. 5. It was serlgically related t ther viruses f the Simbu grup which are currently cnsidered t be arbviruses. athgenicity fr mice, sensitivity t sdium desxychlate, and hemagglutinatin f gse cells are prperties cmmn t many arbviruses. Casals 7 cnsiders that the finding f a serlgical relatinship t an arbvirus

63

64 27- - justifies the inclusin f an agent in the arb "family." A lgical apprach t the classificatin f arb viruses based n antigenic relatinship was first frmulated by Casals.-?,-y As defined by him, distinct viruses that crss-react by ne r several serlgic tests are cnsidered as members f a grup, with the understanding that the degree f verlap can vary frm ne extreme, where the viruses are nearly indistinguishable, t the ther extreme in which it is difficult t demnstrate any relatinship. Viruses are assigned t established grups, r t new grups, n the basis f the reactivity f their hmlgus sera with ther viruses; the reactivity being due t the fact that cmmn antigenic cnstituents are shared by the members f a grup. f the serlgical tests, the HI test with multipleinjectin sera (r ascitic fluids) has been used mst widely fr classificatin purpses. Multiple injectin sera (r AF) are preferred t single-injectin sera since it has been repeatedly shwn that sera frm animals given several injectins f ne virus react with a far wider spectrum f antigens than d sera prduced by a single injectin. The basic value f the HI test fr classifi catin purpses lies in the fact that, in general, it shws a greater width f verlap than either the CF r NT test, and in the ease with which it can be carried ut in large scale. There are cases, hwever, in which the latter tests are mre inclusive. With grup C^and Bunyamwera1 viruses, - fr example, the GF test shws a wider verlap than des the HI.

65 .,

66 28- - With the Simbu grup it appears that the CF test is als mre applicable fr gruping purpses. Fr ne thing, many f the members f this grup have nt been shwn t prduce hemagglutinating antigens, and secndly, the crss reactivity by HI is less than by GF. By GF tests, n the ther hand, there is significant crss-reactivitjr amng several f the agents, and by this methd the relatinship between the Utinga and rpuche viruses wa.s first nted. n the basis f Casals prpsed criteria f classifying a new virus in a given grup, if it crss-reacts with ther members f that grup, the Utinga virus may thus be classified in the Simbu grup. It shuld als be re-emphasized that testing f this new agent by the Belem Labratry against reference grup sera f the ther viruses f that regin failed t shw any antigenic similarities. n the ther hand, the absence f, r very lw-titer crss reactins f Utinga with the ther members f the Simbu grup by HI, GF and NT testing indicates that this new agent is indeed a distinct virus and nt simply a new strain f ne f the ther viruses. The results f the crss-nt tests are admittedly nt cmpletely clear with regard t the gres.ter neutralizing capacity f the Utinga virus by its hmlgus serum as cmpared with the ther sera (r AF). This may be due t the lack f success in prducing a hyperimmune Utinga ascitic fluid with a high neutralizatin index. n the ther hand, the absence f

67

68 29- - crss-neutralizatin between the Utinga and rpuche viruses is in agreement with the results previusly nted by Bensabath. 3 f the 885 human, mammal and bird sera screened fr HI antibdies t the Utinga virus nly 5 sera 1 marsupial, reactins. 1 rdent, 1 human) (2 slths, shwed lw-titer psitive The finding f such lw-titer reactins is in itself nt very cnclusive evidence f actual infectin by an agent (fr reasns that will be discussed belw), and it is essential t btain cnfirmatry evidence by at least ne ther serlgical test, althugh admittedly this is nt always pssible. It is generally accepted that NT antibdies remain detectable fr lng perids, infectin. especially after clinical Less is knwn abut the duratin f HI antibdies, but apparently they persist cnsiderably lnger than F antibdies, and in general the results f HI tests usually agree with thse f the NT tests. u Hwever, when three HI psitive sera were tested by NT tests, the results were at best equivcal. Whereas in that test the hmlgus hyperimmune ascitic fluid neutralized lg LD^q f virus, the 2 slth sera shwed lg neutralizatin indices f nly 1.2 and 21.6 respectively, and the marsupial serum nly.5 r less. As incnclusive as these results are, that these, slth sera; it is interesting at least suggestive reactins, were fund in the slth being the nly animal t date frm

69 '.

70 3- - which the Utinga virus has heen islated. Als, bth f these slths were f the Bradypus tridactilus species. Hwever, it shuld als be nted that ne serum frm a pig (UN 4p) als gave similar incnclusive results. JBme f the factrs that might explain the abve results shuld be cnsidered. It is pssible that the psitive HI reactin may have been due t nn-specific inhibitrs which were nt remved by the acetne treatment f sera, r that these were expressins f heterlgus crss-reactins. Such crss-reactins culd be due t ne f the ther currently knwn Simbu grup viruses, even t an agent that as 3^et has nt been islated. even pssible, althugh admittedly less likely, r It is that such a crss-reactin culd be with agents frm a different arbvirus grup. Whitman and Shpe, 41 and Casals 8 have reprted lw-grade crss-reactins between arbviruses that hithert have been cnsidered as belnging t distinct antigenic grups. If, n the ther hand, we assume fr the mment that the HI reactins were specific, negative, what culd explain the r at best incnclusive, results f the NT tests? It is pssible that animals simply d nt prduce high titers f NT antibdy t Utinga infectins. This pssibil ity is als suggested by the fact that in the crss neutralizatin tests the hmlgus immune ascitic fluid, prduced by several inculatins f virus with adjuvant.

71

72 31- - gave nly 1.6 lgs f neutralizatin. Anther pssibility, which has nt been ruled ut, that either the virus is nt easily neutralizable, "accessry factr" is needed in these reactins. is r that Recently differences have arisen cncerning the rle f the "accessry factr." It was nted by Sabin that neutrali zatin f the muse-adapted dengue virus by intracerebral tests in mice was dependent n tw factrs - a nnspecific, heat-labile accessry substance, is heat-stable and a specific antibdy which (56 C fr 3 min.). Additin f fresh animal serum, which in itself has n neutralizing activity, fully restred the neutralizing capacity f a heated dengueimmune serum. virus, Similar effect have beennnted fr vaccinia ^ and Western equine and St. Luis encephalitis immune sera. 1 Q 2:7 At the same time it appears that at least in sme instances incubatin is mandatry, in rder t reveal a neutralizatin r virtually s, effect.^ reprt n rpuche virus, Andersn _et al p in their nted that neutralizatin f this virus was als markedly enhanced by the additin f fresh serum, i.e., accessry factr. As previusly nted, n accessry factr was emplyed in the present NT tests with Utinga. The pssibility that NT antibdies fr Utinga may decay faster than HI antibdies shuld als be cnsidered, althugh this seems less likely. Althugh it is interesting t speculate abut the significance f the abve results, it

73 ...

74 32- - is my pinin that withut further cnfirmatry evidence these findings cannt be interpreted as indicative f infectin with the Utinga virus. The results f the HI tests with the Belem human sera frm were negative except fr a few, lw-titered reactins. The significance f these lw titered reactins is difficult t interpret. The lack f cnsistent, reprducible results wuld seem t indicate that the bserved reactins were prbably due t nn-specific inhibitrs in the sera, rather than t specific antibdies. The difference in reactivity f these sera with the Belem and YARU antigens cannt be explained at this time. As had been nted in the crss-hi, CF, and NT tests the relatinship f Utinga virus even with rpuche is a limited ne. It is fund nly by the CF test, and appears t be a ne-way reactin, with the Utinga hyperimmune serum reacting with the rpuche virus but nt vice-versa,. Thus, the Belem human sera psitive by CF and NT fr rpuche, did nt react with the Utinga virus. Here again, sme f the factrs mentined, such as use f an accessry factr, may be imprtant in explaining the difference. It thus appears that at least in the species tested, the Utinga virus des nt seem t be widely distributed. The vectr respnsible fr its transmissin has nt yet been identified, and it is pssible that the species which is its main reservir has nt even been tested.

75 .

76 Failure t demnstrate antibdies t this virus in the large number f human febrile cases that were screened suggests that this agent is prbably nt an imprtant factr in human illness in the Belem area.

77

78 34- - summary A newly islated arbvirus, the Utinga virus, was studied t determine sme f its serlgical character istics and epidemilgy. Crss hemagglutinatin-inhibitin, cmplement-fixatin, and neutralizatin tests established that this virus belngs t the Simbu grup f Arbviruses, crss-reacts by CF with the rpuche virus but is distinct frm any f the ther members f this grup. A serlgical survey f 1336 human, mammalian and bird sera frm the Belem regin f Brazil disclsed nly 5 sera with lw-titer psitive HI reactins with the Utinga antigen, but these culd nt be cnfirmed by ther serlgic tests. The absence f antibdies in the human sera indicates that this agent prbably has nt been an imprtant factr in human illness in the Belem regin.

79

80 TABLES and FIGURES

81

82 Figure K Map shwing lcatin f the Belem study areas n the ara and G-uama divers in the State f ara, Brazil Area inclsed within square represents regin frm which all f the sera screened fr antibdies t the Utinga Virus were btained. Shaded area represents trpical rain frest climate zne. (Map after Causey,et al. Am. J. Trp. Med. & H g., J_:228, 1961.)

83 «

84 36- - Figure 2. A hypthetical arrangement f an HI test n a clear Lucite* plate (Cke Engineering Cmpany, Alexandria, Va. ). The results as shwn are read as fllws: 1 = cmplete hemagglutinatin-inhibitin 2 Q - partial hemagglutinatin-inhibitin 3 + = n hemagglutinatin-inhibitin

85

86 FRM 14..EAAQ. VIRUS -DATE. ASSAGE / /-67 SERIAl LQ IN_?AJ^- DILUTIN_ Utihga AMUNT CC RUTE. Jl9i..N.8HA.L_.A SciTtc_ ljiij? IJL 'cf serum. BcAn8H7SS passage - 3 CC. DILUTIN REMARKS 12 -/-«# J.ir.^Ql i7_ mm. Taral " / + /? 6 s f r 4* / / s 3 4 MICE 8 /? S I 7 r T Figure 3. A typical "muse card" used fr recrding data f neutralizatin tests in mice. The hypthetical data shwn is interpreted as fllws: +? S Ttal muse died muse missing frm cage, presumed t have died and been eaten by mther muse shwing bvius evidence f illness number f mice surviving n any particular day

87

88 38 a >5 d vq. H * «r f>5 -H <c3 ni i 1 *H - M <3 s C rh rh!h!h,.. rp - S> 'd Sa rd i! H d s H - S»H p IH rh Si th IH rh ^3 'd <* p 1 b g 1 -H s3 s3 rh1 *H W b <D rh H C THE SIMBU GRU F ARBVIRUSES X d Si 3 H w H d rh S 'd!> c i 1 <3 rh H 9S - H ft a CS.H H d i 1 H bc d rd rsn t-m si bc i 1 H > rh 5>! C d i l rh rh H C rh H H rd rh 4* c rh <3 a X rh d rh v nd <3 rh >: H d cm 'd H d ft S; d Q d rh t> rh t> pq pq H d rh d H s! w d -H»H p rh <m M #s rq S*3 <3 I 4h 1 th M H Q c H t-3 <3 «s >3 IS) p - <m -H i 1 H d «H <3 H 'd H C 9* rh t3 np H fd d H d W rh d EH H d EH L IS C 3 C _ V N Lf nt U C C C C V- ls V rv d 'd t" U C d H d CH <3 C *v rh H H d *\ & rh pq IS IS v V C 5- r "c H pq b H >* r\ H d b H r3 S H nd s M IS IS V C * V C t v c ^ b K CM d H > s K rh K i 1 i 1 r. r <3 Q g > b H cvj i l i l H S) CM ft d k rh d ft si p C H- C bd g rh K s H C p S b C U U IS <3 K S 3h M S

89 -

90 39 lb An 555 San g Simbu Sathuperi rpuche Akabane Ingwavuma v Manzanilla ( H i I fa H - CM H CQ <«s «JU 'T* m fa CM Antigen Units Used CM CM V 6. Utinga 6. M Buttnwillw 6.5 GRSS HI TESTS WITH SIMBU GRU VIRUSES Yaba 7 -M- -M* C T V II 3 r-h i 1 H w cts w EH < a3,w l a5 rh i 1 H c3 N 3 ctf

91 .

92 t- h -H >H hi* C b J h vh <1 CM C Hi* v v T NJ\ pq IA* =3j D- h -H H* la pq C C pq LA <3 C D ph *H njtpq v LA A v T t pq «sd M C W Eh M ja* w M H Q V v 1 C C\J I la EH CQ i t <-< rh h H - r-l C h ph EH C w EH ph h C EH i-q t C pq pq 3 jq 3 ph *H H <J LA C h C 1 3 rd - C -H h h -H <! LA ft 1 3 h -H ft rc! hj* c h v Vj\ Hi* v v v hf' v Ht* v Hjv ' J v LA i A v hj* v \ hj* V LA J \ H* V v LA cm A C J v Hi* v \ v v H* v \ H* v LA cvi \ v T- Hi* v nj* v v nj* H* V v hj* V T V <J* C v LA CVI v LA J \ C J *- v LA J \ J r LA v LA CM v v Ht v LA J \ J t A H- -nt* v Hi* V v r hj* v hj* V s_ J LA hj* V \ J r v Ht CVJ T Hj* v \ v v LA AJ A cvi LAK v LA J v H* v \ C v LA CVI \ J LA Ht* v \ v HiVQ \ C Ht* v \ C v LA J \ V v LA CVJ v LA CVI v LA J \ v LA J V Hi* v, \ C J H* 7s v LA V LA CVJ \ C CVI T~ A v LA J v Hiv C J -* hj* v \ J A Hj- v ih 1 is! h cc3 rh h *H rh <} J -H 1 > Is S h *h b 3 <J J pq pq -Hi 1 h "H -p h <q cm 3 -H pq Is i A{ <q pq h»h < LA r hj* H* v \ v T v LA J C J hj* v C" H* v \ v T~ hj* V \ J LA v LA J 'A. v LA J CD hjv V v LA J H* v \ C 4/64 M t q c±> H!v «u C 3 C t M > hj* v \ H* v v hj* Hj* \ \ CVJ LA Hi* v Hi* Hj- Hf* v v V T CVI JA \ C J CM LA T C pq c±s (H EH ts; <1 pq J M <d rh i 1 H Is pq -p -p 3 pq 3 p» bd pq H i 1 H H 3 ts] pq g ^q 3 ft h LA th H* H h ft ^q -p CQ i J a H C E LA LA LA LA pq <1 pq pq <q <1 rq LH pq th th & (H # Anti-cmplementary h =q LA pin *H LA <t{ LA H LA

93 *

94 41- - TABLE IV CRSS-NT TESTS WITH SIMBU CRU VIRUSES* Immune Ascitic Fluids Buttnwill ^ ) p! p ft rl N S Sh CD, Xi G b H H Li L Li p a rl c C.1 D <3 i Utinga Akabane Buttnwillw.5 Ingwavuma. Manzanilla.5 rpuche.6 Sathuperi S imbu.1 Sang. lb An 555. Yaba 7. 1 H J JH ru Akabane 1.6 I I M S VIRUS Utinga >4.7 2: > * Results expressed as lg neutralizatin index

95

96 42- - TABLE V Surces f Sera Tested fr Antibdies t the Utinga Virus Surce N. Years Tested Represented G-enus & Species Marsupials. T Marmsa sp. Marmsa murina Metachirus nudicaudatus Galurmys sp. Didelphis marsupialis hilander pssum Rdents ,.219 "ST Mnkeys... Cebus Saimiri Sciurus Tamarin Sp Hwler ,. Bradypus tridactilus Chlepus brasiliensis Tamandua sp nt identified Bats u ryzmys geldi rechimys guyannenis ris Nectmys Echimys Cendu Unidentified Slths. N. in Each Year, Birds Humans Unidentified Carllia sp. Urderma bilbatum Artibeus cinereus TS

97 . >> I..,,,,,,,,,,,, r :...

98 43- - TABLE VI Sera Giving sitive HI Reactins with Utinga Antigen (Ttal tested sera) Serum Ed 78 Surce Date btained HI titer* Slth Brad.ypus tridactilus Bel 212 H Man Ed 147 Slth Bradypus tridactilus Ma 199 An Marsupial Calurmys sp R 7242 An 1287 Rdent^ Cendn sp * HI titer expressed as reciprcal f highest dilutin giving cmplete inhibitin.

99

100 44 - tp s. in testing N 196 ia be h a r-f p bc a H h I d v d c - rh C CD *H a <t{ C <D d EH bcj a Jx, H H H > d d m d EH <- V H v a d v i p a a be h a H h a d C rq H a a pq d d a d a a H d d v d v d d Q <3 d M <+H p a I C a p a a p c ' d a pq a H a C5 d H'1' -I* d.» a i>^ d H h cc3 ct> d d C a a a c>3 p a d J bc a >H h a -h 3 M d 53 i j a a -! i a a b b H.H a a t-l d d d <3 3>>!>H d^ p p a b b a a p H *H p p bc d d a d d J cc5 tq a p p V c d u ctf a (D C a C c a a a j a a d p i i

101

102 45- - vl. '1* V^. V bc p p fcdv p^ -p HN CQ A V JV -3- -ia-^t v cm v la [pra W' ', *» > Eh H H EH I bc >- ftp! VQ I N raf immn A I p b p! KNKNKNKN -- EH bft> M «H cm H «d I W I =; b H <s\ cv3 H KN < KN bd > bd b!> bd p ra p p H > I CQ H pq <3 Eh p K * x> C Eh rd p E C r ra p p p a ra C EH < j ftp! p bc 1 p V p bd n ra <J CQ M > bc I V Eh ftp! V bn ICVVI I C V I ft> b <J bc!h-p< p> 1!! I I I I C -4* CM CM I J I! I I I ra I I H H p p «i i ra p r ja p p r f p i i p np np p p p p p a i I m l b p p bc <lj e i I V I J CM CM J C i C *r~\ W CD < rp bdp! p >3 b b rap a p p Dh V t * i r t LA LA V V V V VQVQVVVVVVVVVV nnnnnnnnnnnn Q i! H p p p. p p p p p!,p p --pra H adaap bh b b p i I i 1 Cl5 C gffippshfflnfm Cli r p! d > 1M ra ra > p ra p p i EH 'I4 CM LA LA C LA C N N N K- NC a a a <x> -a- n-p kn t>-'- -N'-<J*-^ J_J t I t_j > pq pq pq pq E<q E3 w i i p p ft p 4 d»!>;, i I i! Cd ra h Eh Eh Ep p I p C C bp a p 3 #r«l I_I p ip ra i i ra p, p M p p p, p ft> ) l >

103 «: :.

104 46- REFERENCES 1. Andersn, C.R., Spence, L.T., Dwns, W.G., Aitken,T.H.G, "Manzanilla Virus: A New Virus Islated frm the Bld f a Hwler Mnkey in Trinidad, W.I." Amer. J. Trp. Med. & Hyg., 9:73-8, i Andersn, C.F., Spence, L., Dwns,.. and Aitken, T. H.G., rpuche Virus: A new human disease agent frm Trinidad, W.I. Am. J. Trp. Med. & Hyg., 4: , Bensabath, G-., Belem Virus Labratry, persnal c mmunicatin. 4. Casals, J., Cmplement-fixatin test fr diagnsis f human viral encephalitides. J. Immunl., 56: t Casals, J., Viruses: the versatile parasites, the arthrpd-brne grup f animal viruses. Trans. N.Y. Acad. Sci., Series II, 19: , Casals, J., Antigenic classificatin f arthrpd-brne viruses. rc. VI Internat. Cngr. Trp. Med, and Malaria, 5T34-47, Casals, J., rcedures fr Identificatin f arthrpdbrne viruses. Bull. Wrld Health Qrg., 24: , Casals, J., New develpments in the classificatin f arthrpd-brne animal viruses. rc. VII Internat. Cngr. Tr. Med, and Malaria, 11:13-34, Casals, J., and Brwn, L, V., Hemmagglutinatin with arthrpd-brne viruses. J. Exper. Med., 99: Casals, J., and Whitman, L., A new antigenic grup f arthrpd-brne viruses: the Bunyamwera Grup. Am. J. Med, and Hyg., 9:73-77, i Casals, J. and Whitman, L., Grup C, a new serlgic grup f hithert undescribed arthrpd-brne viruses, immunlgical studies. Am. J. Trp. Med. & Hyg.. 1: , 1961

105

106 Causey,.R., Causey, C. E., Marja,.M. and Maced, D. G-., The islatin f arthrpd-brne viruses, including members f tw hithert undescribed serlgical grups, in the Amazn regin f Brazil. Am. J. Trp. Med. & Hyg., J_: , Causey, C. E. and Causey,. R., The Arthrpd-brne viruses f Brazil in relatin t wrld grups. Revista d Servic Expecial de Saude ublica, 12:9-13* "19^2 14. Causey,. R., The islatin f virus frm natural and sentinel hsts in the Amazn Valley. Revista d Servic Especial de Saude ublica, 12;25-31, Clarke, D.H. and Casals, J., Techniques fr hemagglutina tin and hemagglutinatin-inhibitin with arthrpdbrne viruses. Am. J. Trp. Med. & Hyg., 7: , Fultn, F. and Dumbell, K.R., The serlgical cmparisn f strains f influenza virus. J. G-en. Micrbil., 3:97-111, Herrman, E.C., Jr. and Engle, C., Tumr cell-induced muse ascites fluid as a surce f viral antibdies. rc. Sc. Exp. Bil. & Med., 98: , Hrsfall, F.L. Jr. and Tamm, I.(Ed.), Viral and Rickettsial Infectins f Man (4th ed.), J. B. Lippinctt C., hiladelphia, Lennette, E.H. and Schmidt, N.J.(Ed), Dia.gnstic rcedures fr Viral and Rickettsial Diseases/ 3rd Ed., Am. ublic Health Assc., Inc., Hew Yrk, McCarthy, K. and G-ermer, W.D., Tw heat-labile factrs in nrmal sera which neutralize varila virus. Brit. J. Exper. ath., 3.3: , ya, A., kun, T., gata, T., Kbayashi, I. and Matsuyama, T., Akabane, a new virus islated in Japan. Jap. J. Med. Sci. & Bil., 1^4:11-18, inheir, F., inheir, M. Bensabath, G-., Causey,.R. and Shpe, R.E., Epidemi de Virus rpuche em Belem. Revista d Servic Especial de Saude ublica, 12:15-23, T9 62. ' 23. rterfield, J.S., Use f gse cells in haemagglutinatin tests with arthrpd-brne viruses. Nature, 18:121-, 1957.

107 * ' <* - «" ' <. 5,, *.., 4 4 * * t. t *..,.

108 4824. rterfield, J.S. and Rwe, C.E., Hemagglutinatin with arthrpd-brne viruses and its inhibitin by certain phsphlipids. Virlgy, JJ_: , i Reed, L.J. and Muench, H., A simple methd f estimating fifty per cent end pints. Am. J. Hyg., 27g * Sabin, A. B., The Dengue grup f viruses and its family relatinship. Bact. Rev., ht: , Sartrelli, A.G., Fischer, D.S, and Dwns, W.G., Use f Sarcma 18/TG t prepare hyperimmune ascitic fluid in the muse. J. Immunl., 96: , Sever. J. L., Applicatin f a micrtechnique t viral serlgical investigatins. J. Immunl., 88:32-329, Shpe, R. E., The use f a micr hemagglutinatininhibitin test t fllw antibdy respnse after arthrpd-brne virus infectin in a cmmunity f frest animals. Anais de Micr., Vl, 11: , Shpe, R.E., The serlgical identificatin f arthrpdbrne viruses. Revista d Servic Especial de Saude ublica, jjh33-3^7 T Simpsn, D.I.H. and Williams, M.G., Identificatin f viruses islated in Nigeria. East African Virus Research Institute Reprt - July i~96'3 t December RepriT?1.?r32f347 "l9^5'. " 32. Taylr, R.N.(Ed), Arthrpd-Brne Virus Catalgue f the Wrld, Sathuperi #427 U. S. Gvernment rinting ffice, 1967? (in press). 33. Ibid., 34. Ibid., Buttnwillw # Theiler, M., Studies n the actin f Yellw Fever virus in mice. Am. J. Trp. Med., 24: , Theiler, M., Actin f sdium desxychlate 11 arthrpdbrne viruses. rc. Sc. Exper. Bil. & Med., 96: , ~ 37. Theiler, M. and Dwns, W. G., rpuche: the stry f a new virus. Yale Scientific Mag., March, 37:1, Ingwavuma #126.

109 * * t. t,

110 Tikasingh, E.S., Spence, I. and Dwns, W. G-., The use f adjuvant and Sarcma 18 cells in the prductin f muse hyperimmune ascitic fluids t arbviruses. Am. J, Tr~. Med & Hyp;., J_5: , Weinbren, M.., Heymann, C.S., Kkernt, R.H. and atersn, H.E., Studies n arthrpd-brne viruses f Tngaland. VII. Simbu virus, a hithert unknwn agent islated frm Aedes (Banksinella)circumulutelus. Suth African J. Med. Sci.,,22:93-12, Whitman, L. and Casals, J., The G-uama G-rup: a new serlgical grup f hithert undescribed viruses, immunlgical studies. Am. J. Trp. Med & Hyg., 1: , Whitman, L. and Shpe, R.E., The Califrnia cmplex f arthrpd-brne viruses and its relatinship t the Buyamwera grup thrugh G-uara virus. Am. J. Tru. Med. & Hyg., _M:691-96, Wdall, J.., Directr, Belem Virus Labratry, ersnal cmmunicatin. 43. Wdall, J.., Directr, Belem Virus Labratry, ersnal cmmunicatin. 44. Unpublished results in 1965 Annual Reprt f University f Ibadan Arbvirus Research rject, p*. 35.

111 . 4 t *. «*.... '

112 *

113

114 YALE MEDICAL LIBRARY Manuscript Theses Unpublished theses submitted fr the Master's and Dctr's degrees and depsited in the Yale Medical Library are t be used nly with due regard t the rights f the authrs. Bibligraphical references may be nted, but passages must nt be cpied withut permissin f the authrs, and withut prper credit being given in subsequent written r published wrk. This thesis by I~kr etchary has been used by the fllwing persns, whse signatures attest their acceptance f the abve restrictins. NAME AND ADDRESS f) / " A-. DATE mj \rnll D/j^tr /U ^ /">

115