Am. J. Trap. Med. Hyg., 32(5), 1983,pp. 1164-1171 Copyright @ 1983 by The American Society oí Tropical Medicine and Hygiene

CHARACTERIZATION OF EIGHT NEW PHLEBOTOMUS FEVER SEROGROUP ARBOVIRUSES (BUNY AVIRIDAE: PHLEBOVIRUS) FROM THE AMAZON REGION OF BRAZIL*

AMELIA P. A. TRAVASSOSDA ROSA,t ROBERTB. TESH,:I: FRANCISCOP. PINHEIRO,t JORGEF. S. TRAV ASSOS DA ROSA,t ANDNORMAN E. PETERSON§ tlnstituto Evandro Chagas,Fundação Servicos de Saúde Pública, Ministerio da Saúde, CP 621, 66.000 Belém, Pará, Brazil, :l:YaleArbovirus Research Unit, Department of Epidemiology and Public H8I!lth, Vale University School of Medicine, P. O. Box 3333, New Haven, Connecticut 06510, and §U.S. Army Medical Research Unit, Belém, Pará, Brazil

Abstract. Eight new members of the phlebotomus rever arbovirus serogroup(family Bun- yaviridae; genus Phlebovirus) from the Amazon region of Brazil are described. One serotype was recovered from a febrile patient, three from smaII wild animais and falir from sand mes. A smaII serum survey carried out with the human isolare, Alenquer virus, suggeststhat it rarely infects mano Complement-fixation and plaque reduction neutraIization testswere don~ comparing the eight new viruses with other members of the phlebotomus rever serogroup. A close antigenic relationship was demonstrated between one of the new agents (Belterra) and Rift VaIley rever virus. This finding is of considerable interest and deservesfurther investi- gation. Addition of these eight new viroses to the genus Phlebovirus brings to 14the number of serotypes known to occur in the Amazon region and to 36 the total number reportedworld- wide. More detailed clinicaI and epidemiologicaI studies should be conducted in Amazonia in arder to define the public heaIth impact caused by phleboviruses.

During the early 1970s construction began on ver serogroup (family Bunyaviridae; genus Phle- an extensive network of highways acrossvast areas bovirus). The purpose of this report is to of virgin tropical forest in the Amazon region of characterize the eight new phlebovirus serotypes, Brazil. As these highways were completed, colo- to describe their antigenic relationship to other nists began to settle in accessibleland adjacent to members of the phlebotomus fever group and to the new roads. Most of the settlers were from out- discuss their potential role in human disease. side the region, and became engaged in agricul- tural activities. Because of their presumed non- MATERIALS AND METHODS immune status and their close contact with the Viroses forest, it was important to determine which local pathogens, including arboviruses, were potential- The .56phleboviruses used in thisstudy are list- ly hazardous to their health. Consequently, stud- ed in Table 1. The eight new Brazilian serotypes ies were conducted at numerous sites along the are V rucuri, Itaituba, Alenquer, Turuna, Belter- highways to determine the possible occurrence of ra, Joa, Oriximina and Munguba. The circum- arboviruses and to assesstheir public health im- stancesof their isolation are given in Table 2. The portance. As a result of these investigations, a approximate locality in which they were recovered number of arboviruses (some known and others is shown in Figure 1. new to science)were isolated. Among the new vi- Stocks of 35 oí the viruses were prepared from ruges were eight members of the phlebotomus fe- infected mouse brain or Vero cells. These stocks were used in plaque reduction neutralization tests and to inoculate newborn mice for preparation of Accepted 4 January 1983. antigens. We were unable to work with Rift Val- ley rever (RVF) virus, since it is a restricted agent * Address reprint requests to: Dr. Robert B. Tesh, in the Vnited States. However, Dr. C. J. Peters, Vale Arbovirus Research Unit, Department oí Epide- V.S. Army Medical ResearchInstitute of Infec- miology and Public Health, Vale University School oí Medicine, P.O. Box 3333, New Haven, Connecticut tious Diseases,Frederick, Maryland, kindly per- 06510. formed neutralization tests with this agent and 1164

Vd~:::> CHARACTERIZATION OF NEW BRAZlUAN PHLEBOVIRUSES) 1165

TABLE 1 -' ,- , SURINAME Viruses used in complement-fixation and neutralization teses (OLOM"A Viros serotype' '.'." Strain " N .o Aguacate VP-175A Alenquer Be H 301101 Anhanga Be An 46852 " j'-" Arumowot Ar 1284-64

"RU°-..,_oI ,-' ,-' I "I Belterra Be An 356637 -..,- -Z o' -I .1 ; Buooaventura Co Ar 3319 BOlIVIA--'-._'~ ) '-,. ~ r o__J Bujaru Be An 47693 ~ u I Cacao VP-437R ,o (UIA';"'" . .SITES.SITESO'O, (OlLECT.,.COLLECTKJN , 0-0_0--0_oo_oooJ' Caimito VP-488A Candiru Be H 22511 FIGURE 1. Map of the Amazon region of Brazil, Chagres JW 10 showing study sites and localities where the eight new Chilibre VP-118D phleboviruses were isolated. Frijoles VP-161A Gabek Forest Sud An 754-61 Gordil Dak An B 496d Icoaraci Be An 24262 ISSo Phlo 18* ISSoPhl. 18 Immune reagents ltaituba Be An 213452 The RVF antiserum was made in a sheepand ltaporanga Original Joa Be Ar 371637 was supplied to us by Dr. C. J. Peters. Specific Karimabad I-58 hyperimmune ascitic fiuids against each of the Munguba Be Ar 389707 other 35 viruses were prepared in mice. Infected Naples (Sandt1yrever) Naples BHK-21 cells were used as the immunizing anti- Nique Nique-9C Oriximina Be An 385309 gen for Cacao virus. Ten percent suspensionsof Pacui Be An 27326 infected mouse brain in phosphate-buffered sa- Punta Toro D-40210 A liDe, pH 7.2, were the antigens used to prepare Rift Valley rever Entebbe the remaining immune. reagents. The immuniza- Rio Grande TBM4-719 Saint Floris DAK An B 512 tion schedule for the mice consisted of four intra- Salehabad 1-81 peritoneal injections given at weekly intervals. Sicilian (Sandt1yrever) Sicilian Immunizing antigens were mixed with equal vol- Tehran 1-47 umes of Freund's complete adjuvant just prior to Toscana ISSo Phlo 3 Turuna Be Ar 352492 inoculation. Sarcoma 180 cells were also given in- Urucuri Be An 100049 traperitoneally with the final injection in order to .ISSo Phl. 18 was supplied by Dr. PaoIa Verani, Istitulo Superiore di induce ascitesformation. Sanita, Rome. Its inclusion here is not intended to constitute priority of publication. Complement-fixation tests

provided us with RVF immune serum and inac- Complement fixation (CF) tests were done ac- tivated antigen. cording to a microtechnique modÍfied from Fulton and Dumbell,3 using two full units of guinea pig complementoTiters were recorded as the highest Antigens dilutions giving 3+ or 4+ fixation of complement The RVF antigen used in complement-fixation on a scale of O to 4+. (CF) testswas infected mouse liver which had been beta-propiolactone-inactivated and sucrose ace- N eutralization tests tone-extracted.l The complement-fixing antigens used for Chilibre and Cacao viroses were pre- Plaque reduction neutralization tests (PRNT) pared from infected Vero cells.2The remaining 33 were performed in microplatecultures of Vero cells, viral antigens were made from infected newborn using a fixed virus inoculum (40-150 plaque-form- mouse brain. These were prepared as 10% crude ing units) against varying antiserum dilutions. Af- brain suspensionsin veronal'-buffered saline, or by ter heat inactivation (56°C for 30 min), antisera the sucroseacetone extraction method.l were prepared in twofold serial dilutions (begin- 1166 TRAVASSOSDA ROSAET AL.

TABLE 2 Source, date and geographical area of isolation ofeight new phlebovirusesfrom Brazil , Virusserotype Strain Source Date of isolation Geograpbical area,' Urucuri* Be An 100049 Proechimys guyannensis Apr 19, 1966 Utinga forest, Belém ' (blood)t ltaituba Be An 213452 Didelphis m. marsupialis Dec 7, 1971 Tapacurazinho stream, (blood) ltaituba Alenquer Be H 301101 Human blood May 31, 1976 Ramal das Pias Alenquer Turunl:l: Be Ar 352492 Lutzomyia sp. Jul 20-26, 1978 Km 4, Cachoeira (human bait) Porteira, Oriximina Belterra Be An 356637 Proechimys longicaudatus Sep 22, 1978 Belterra, Santarem (pooled viscera) Joa Be Ar 371637 Lutzomyia spp. Mar 29, 1979 Joa, Altamira (tree buttress collection) Oriximina Be Ar 385309 Lutzomyia spp. Jun 26-30, 1980 Saracazinho, Porto (light trap) Trombetas, Oriximina Munguba Be Ar 389707 Lutzomyia umbratilis Sep 20, 1980 Monte Dourado, Jari (tree buttress Almerim collection) .Six additional strains of Urucuri virus were isolated from Proechimys captured in Utinga forest (4), at Serra do Navio, Amapa (1), and at Porto Trombetas (1). t Sample positivo or method of collection. j: Two additional isolares of Turuna virus were made from sand fties at Porto Trombetas.

ning at 1:10)in phosphate-buffered saline, pH 7.2, cording to their closestantigenic relatives. By CF containing 0.5% gelatin. An appropriate amount test, each of the eight new Brazilian viruses is of virus was tben added to each dilution. Anti- antigenically related to one or more of the existing serum-virus mixtures were incubated overnight at phlebovirus serotypes.In fact, some of theseagents 5°C prior to inoculation. Two microplate wells are indistinguishable by CF testo were inoculated witb each antiserum dilution. The In an attempt to differentiate these a.ntigenically highest antiserum dilution producing ~90% plaque related phleboviruses, PRNT were done with 20 inhibition was recorded as tbe endpoint. selected viruses and antisera. Results are sum- Mouse neutralization tests (MNT) were done in marized in Tables 4 and 5. By PRNT, Munguba suckling mice, using a final serum dilution of 1:4. and Bujaru viruses were clearly separable. Like- Serum-virus mixtures were incubated for 1 hour wise, RVF, Belterra and Icoaraci viruses were at 37°C and tben were inoculated intracerebrally. distinct. Joa and Frijoles viruses were algOdiffer- entiated by this method. H emagglutination-inhibition tests The antigenic relationship between Oriximina, Itaituba, Alenquer, Nique, Candiru, Turuna, Hemagglutination-inhibition (HI) tests were Punta Toro and Buenaventura is more complex performed as described by Clarke and CasaIs,. than the others. By CF test, some of these agents using a microtechnique; serum or plasma was ace- are indistinguishable (Table 3). Most of them are tone extracted. algo related by PRNT (Tables 4 and 5); however, a fourfold or greater difference in neutralizing an- RESULTS tibody titers was demonstrated between the ho- mologousand heterologousviroses, indicating that Table 3 summarizes results of cross-CF tests each of these agents representsa distinct phlebo- between Urucuri, ltaituba, Alenquer, Turuna, vírus serotype. Belterra, Joa, Munguba, Oriximina and the other Toscana, Tehran and Naples viroses were algO known phleboviruses. In con~tructing this table, differentiated by PRNT, as were Salehabad and an attempt was made to group the viruses ac- ISS. Phl. 18 viruses (Table 5).

Joa CHARACTERIZATION OF NEW BRAZILIAN PHLEBOVIRUSES 1167

The eight new Brazilian phleboviruses were ini- number of serotypes in the group to 36. The an- tialIy isolated in newborn mice. AlI isolates kilIed tigenic relationship among the 36 viroses varies suckling mice when inoculated intracerebralIyj considerably. For example, Sicilian sandfly rever average survival time at the fourth passage leveI virus is unrelated to any of the known phlebovi- varied from 3.0 to 6.8 days. With the exception ruges by CF testoIts inclusion in the gentisis based of Turuna virus which rarely kilIed, each of the on a weak relationship demonstrated by HI and new viroses was also lethal by the intraperitoneal fluorescent antibody tests.4,5 Other viruses, such route. In weanling mice, however, only Itaituba as Naples, Tehran and Toscana, are indistin- virus was lethal by the intracerebral routej none guishable by CF test but are distinct by neutral- of the viroses kilIed weanling mice when injected ization method (Tables 3 and 5). Still others, such intraperitoneally. A hemagglutinin active against as ltaituba, Nique, Candiru and Oriximina are gooseerythrocytes was obtained from mousebrains closely related by both CF and PRNT (Tables 3 infected with Urucuri, Joa and Belterra viruses and 4). This complex antigenic relationship ob- after treatment with sucrose-acetone,but identical servedamong phlebovirusesmay be related to their procedures failed to demonstrarea hemagglutinin segmented genome, since the CF and neutraliza- for the other tive new serotypes. tion determinants on the virion are thought to be During our field studies, a total of sevenstrains coded for by different genes.6 of U rucuri virus and three of Turuna were re- The antigenic relationship demonstrated be- covered, whereas only a single isolation of each of tween RVF, Belterra and Icoaraci viruses (Tables the other six agents was made (Table 2). AlI of 3 and 4) is very interesting. This relationship has the Urucuri recoveries were made from spiny rats subsequently been confirmed by HI test, immu- of the gentis Proechimys. A variety of animal sera nofluorescence and enzyme-linked immunosor- from the Utinga forest area were screened by HI bent assay (Dr. J. M. Meegan, Yale Arbovirus test against Urucuri antigen. The resultsare shown Research Unit, personal communication). The in Table 6. The highest prevalence of antibodies close antigenic relationship between these three was found in rodents (Proechimys)and edentates, agents raises an important question of whether although the sample of the latter was rather small. immunity to one would provide cross-protection These serological results, as welI as the seven re- against either of the other two. The answer to this coveries of Urucuri virus from Proechimys, indi- question is relevant, since there is currently con- cate that spiny rats play a role in the ecology of siderable interest in developing a safe and effec- this agent. tive vaccine against RVF virus. It is possible that Alenquer virus was recovered from the blood Belterra virus could be used in domestic animais of a 27-year-old male forest worker. This individ- as a vaccine, or that a non-pathogenic but im- ual had fever, headache and myalgia of 2 days' munogenic recombinant could be formed from duration. His recovery was uneventful, and hos- segments of RVF and Belterra viroses. Further pitalization was not required. A seroconversionto study in this arfa would seemappropriate. Alenquer virus was demonstrated by MNT on Results of the CF test (Table 3) indicate the acute and convalescentsera from the patient. existence of sevenantigenic complexeswithin the In order to get some indication of the frequency phlebotomus rever serogroup. These are as fol- of human infection with Alenquer virus in the lows: 1) a Bujaru complex consisting of Bujaru, Amazon region, sera from 227 selected residents Aguacate and Munguba viruses; 2) a Rift Valley of the region were screened by MNT against the rever complex comprised of RVF, Belterra and virus. Only one of the specimensneutralized Alen- Icoaraci viruses; 3) a Candiro complex consisting quer virus, suggesting that the frequency of hu- of Candiru, ltaituba, Alenquer, Nique, Turuna, man infection with this agent is low. Oriximina, Punia Toro and Buenaventura virus- es; 4) a Naples complex comprised of Naples, DISCUSSION Tehran, Toscana and Karimabad viruses; 5) a Frijoles complex consisting of Frijoles and Joa vi- Results of our study (Tables 3-5) indicare that ruges; 6) a Salehabad complex comprised of Sa- Urucuri, Alenquer, Itaituba, Turuna, Belterra, lehabad and ISS. Phl. 18 viruses; and 7)a Chilibre Joa, Munguba and Oriximina viruses represent complex consisting of Chilibre and Cacao viroses. new serotypes in the phlebotomus fever group. Undoubtedly, additional members will be added The addition of these eight agentsbrings the total~ to this list as new phleboviruses are isolated. 1168 TRAVASSOS DA ROSA ET AL.

TABLE 3 Resw/1sti , ,- j\%IJtimoluis willl36 ,hlcbot usfcvcr "OU, vi...s serot".s Ao"'"'"'" ANTlGEN A~U BUJ MUNRVF BTA ICO CHG COR RG !TA ALE N1Q CDllTUAOax PT

256" Aguacate -O O -O O O O O O O O O O O O 128 .8 256 256 Bu)aru 00 O O O O O O O 000 32 128 128 16 128 256 8 Munguba ---O O O -O O O O O O O O O 128 8 128 8 .8 128 512 64 8 8 8 32 Ri/t Valley rever O O O O O O O O 16 256 256 128 16 8 8 32 2560 512 32 Belterra O O O O --O O O O O O O O 0- 640 512 32 i . O O O 512 1280 coarac! O --O O O O O O O O O O 512 640 256 Chagres 000-00-000000000 128 .

256 Gordil O O O -O O O -O O O O O O O O 32 256 Rio Grande O O O -O O O O -O O O O O O () 8 itaituha O O O -O O O O O ill O ..!!!. ill ill ~ O 128 128 128 32 32 256 256 64 Alenquer O O 0- Ó O O O O O --O -O O 32 8 4 .32 64 256 8 256 16 Nlque O O O -O O O O O O 888488 .256 16 256 64 64 Candlru O O O-O O O O 0-0 0 128 128 128 128 128 64 32 128 64 256 8 16 Turuna O O O -" O O O O O 32 32 32 32 32 8 8

Oriximina O O O O O O O O O ill..!!!. ill ill ~ ill J!!. 128 128 128 -128 128 128 32 PunIa Toro O O O -O O O O O O O -O32 -O16 -256 128 128 128

Buenaventura O O () -O O O O O O O O O -O8 - 128 32 128 16 Toscana O O O --O 8 O O O O O O O . O O O

Tehran O O Q -O O O O O O O O O O 0;0

Naples O O, O ~ O O O O O O O O O O 00

Karimabad O O O -O O O O O O O O. O O 0.0

U .8 O 8 rucun O () O --O O O O O O -O 0",0 8 8 c.. Pacui O O O --!- O O O O O O O O O 00 32 8 ItaporangaO O 0-- O O O O O O O O O 00 8

Sicilian \000-'0 O 00 O 00 00000 " Anhanga" 0'0 O -O O O O O O O O O O O O Frijoles O O O -O O O O O O O O O O O O

Joa O O O O' O O O O O O O O O O O O , Cacao O O O -O O O O O O O O O O O O

Chilibre O O O -O O O O O O 00 O O O O

Caimito O O O -O O O O O O O O O O O O

Saint Floris O () O -li O O O O O O O O O O O

Arumowot O O O .,- O O', O O O O O O OC O O O

Gabek Forest O O O -,. O O 'O r o o o o o o o o

Salehabad o o o -O o o o o O O o o O o o ; ';

.Recip,oc", of higbut ~ti~m diiutioolhigbut ~ti..n dilution o -<414 CHARACTERIZADON OF NEW BRAZIUAN PHLEBOVIRUSES' 1169

TABLE3 Collli"...d J..:; AntiMN'" SUE TOS TEU S"" KAR UaII PAC ITP SFS ANH FRI JOA CAC CIO CAI ~ Awr GF SAL 155 8 O -O O O O O O O O O O O O O O O O O - 8 O O O O O O O O O O O O O O O O O O O -

O O O 09 O Q O O ! 00 O O O O O Ô O () 8 \ O O O O O O O O O O O O O O O O O O O -

O O O O O O O O O O O O 00 O O O O O 8 O 8" O O O O O O O O O O O O O O O O O;;

O O O O O O 00 O O O O O O l) O O O O ê

O O O O O O O O O O O O O O O O O O O Ó

O O O O O O O O O O O O O O O; O Ó; O O '-,

) O O O O O O O O O 000 O O O O O O 0;-

O O O O O O O O O O O O O O O O O O O - "" ,,:,";:: O O O O O O O O O O O O O O O O O O Q~ , 0000000000000000000"", O O O O O O O O O O O 6; O O Q O O O O '- O O Q O O O O O O O O O O O O O. O O O - 16: - -O O O O O O O O O O O O' O O 6 O O O 128 256 -O O O O O O O O O Q O O O O O c-0 O O - 128 , O _25628 _ 25628 _ 6428 O Q O O O O 0- Q O O O O O O O O 1 1 1 . 128 256 64 O 32 128 128 O O O O O O O O O O O O O O O -,,- 256 256 256 . O ---O32 32 32, O O O O O Q O O O O; ,. O O O O C'i'" II 32 128 32 32 0- ---O O O O O O O O O O O O O 0.""- 8 8 8 128 8 64 O -O O O -O O O O O 00 O O 0000"'- 8 32 16 256 O -O O O O -O O O O O O O O O O O I) """" 8 32 ..

O O O O O O O ~ O O O O O O O O O O Ó 2' ...8 256 {,; O 0,1) 0,,\ O O O O 32 O O 9" O O ~ O O O O., "írtil

" 256 O -O O Q O O O O O -O O O O O O O O O -,,- 8 O O O O O O Q ,O O O ~ ~ O O O O O O O - 32 8 256 32 O O O O O O G O, O O --O O O O O Q 04", 128 32 """ O O O O O O 'O O '6 O O O ~ ~ O O O O Ó ~l ; 128 32 256 O O O O O O O O O O O O O -O O O O O, ,'" 128 O O O O O O O O ,O O O O O O ~ O O O O 8 O O O O O O O O ~'O O O O O O ~ O O O " 32 .0 O O O O O O O O " ~ Ó O Q O O O!:J!! O 0- 128

f} O O O O O O O O 00 O O O O O O~O J:!!. '. 128 4

O O O' O O O O O I) li O O O O O O O O ~ ~ 128 128 128 256".;i";:;"",r,,;i~'7":~ -O M 256 1170 TRAVASSOS DA ROSA ET AL.

TABLE 4 Results ofplaque reduction neutralization tests with selectedphlebotomus fever group viruses

AntiJerum Virus BUI MUN RVF BTA ICO ITA ALE NIQ CDU TUA ORX MungubaBujaruRift Valley rever 1,280*<1080 5,120<10160 320 640 40 <10 <10~ <10 -<10 <10

IcoaraciIWtubaBelterra --<10 <1032010 10,2402,560<10 5,120 <10 640 640 <10 320

Alenquer <10 80 <10 <10 <10 <10 Nique <10 <10 80 40 20 <10 Candiru <10 <10 <10 1,280 10 <10 Turuna <10 <10 40 320 1,280 40 Oriximina 160 20 160 320 20 160 .Reciproca! of highesl antiserum dilution producing ~90% piaque inhibition. -, nol tested

TABLE 5 Results of plaque reduction neutralization tests with selected phlebotomus fever group vinlses

Antiserum Viruo PT BUE TOS TER SFN FRI ]OA SAL ISS

TehranNaplesBuenaventuraToscanaPuntaFrijoles Toro 1,280*--1020--~5,120 40 20 <1080 3204010 5,120 1,280 --

Joa 320 10,~40 -- Salehabad 320 <10 ISS. Phl. 18 <10 160

.Reciproca! of higheol antiserum dilution producing >90% plaque inhibition. -, nol testro.

The identification of eight new serotypesbrings may be related to its rich sand fly1 and vertebrate the total number of phleboviruses known to occur fauna. Only two of the 14 viroses (Alenquer and in the Amazon region of Brazil to 14. The large Candiru) have been recovered from humans. The number of virus serotypes present in the region illness associatedwith Alenquer and Candiru vi- rus infection is similar to the disease (pappataci or sandfly rever) produced by most other phlebov- TABLE 6 iruses which infect humans.4.8-10In view of the Prevalence ofhemagglutination-inhibiting antibodies to fact that Alenquer and Candiru viroses each have Urucuri virus in various wild animal SeTafrom the been recovered only once from man and that an- Amazon region tibody rates to them among human residents of No. positive/total % the Amazon region are low,4 it appears that these Animal type seratested positivo two agents infrequently infect humans and thus Rodents are of little public health importance. Nonethe- Proechimys 144/1,253 11.5 less, it is important to continue surveillance of Others 3/1,130 0.3 Marsupiais 0/983 O human diseaseassociated with these agents, es- Primares 1/136 0.7 pecially among colonists moving into new geo- Edentates 1/8 12.5 graphic arfas. In addition, more extensive sero- Chiroptera 0/203 O logical surveys with alI 14 phlebovirus serotypes Ungulates 0/4 .O should be done to properly assess their public Birds 1/2,652 <0.1 , health importance. CHARACTERIZATIONOF NEW BRAZILIAN PHLEBOVIRUSES' 1171

ACKNOWLEDGMENTS hibition with arthropod-borne viroses. Am. J. Trap. Med. Hyg., 7: 561-573. The studies in Brazil were funded by the Pro- 2. Tesh, R. B., Chaniotis, B. N., Peralta, P. H., and gramma dosPolos Agro-Pecuarios da Amazonia Johnson, K. M., 1974. Ecology of viroses iso- (POLAMAZONIA), Superintendencia de Desen- lated from Panamanian phlebotomine sandflies. Am. J. Trap. Med. Hyg., 23: 258-269. volvimento da Amazonia (SUDAM), Ministerio do 3. Fulton, F., and Dumbell, K. R., 1946. The sero- Interior, Brazilian Government. logical comparison of strains of influenza virus. Part of this work was carried out at the Vale J. Gen. Microbiol., 3: 97-111. Arbovirus Research Unit while A.P.A.T. and 4. Berge, T. O., 1975. International Catalogue ofAr- ].F.;:>.T. were in residence as Visiting Scientists, bO'lliruses. DHEW Publication No. (CDC) 75- 8301, U. S. Department of Health, Education, supported by fellowships froro the Pan American and Welfare, p. 789. Health Organization. The study at Vale was algO 5. Tesh, R. B., Peters, C. J., and Meegan, J. M., funded by grants from the National Institutes of 1982. Studies on the antigenic relationship among Health (AI-IO984-10), from the U. S. Army Med- phleboviruses. Am. J. Trap. Med. Hyg., 31: 149- 155. ical Research and Development Command 6. Bishop, D. H. L., and Shope, R. E., 1979. Pages (DAMD 17-81-C-1121)and from the Office ofNa- 1-156 in H. Fraenkel-Conrat and R. R. Wagner, val Research(N14-78-C-OIO14). Urucuri vírus was eds., Comprehensive Virology, VaI. 14. Plenum isolated during a collaborative study between the Press, New York. Evandro Chagas Institute (IEC) and the Rocke- 7. Martins, A. V., Williams, P., and Falcao, A. L., 1978. American Sand Flies (DiPtera: Psycho- feller Foundation. didae, Phlebotominae). Academia Brasileira de The authors wish to thank Dr. C. ]. Peters for Ciencias, Rio de Janeiro, pp. 195. providing the RVF antigen and antiserum. We are 8. Bartelloni, P. J., and Tesh, R. B., 1976. Clinical algOgrateful to Dr. ]. M. Meegan for performing and serological responses of volunteers infected with Phlebotomusrever virus (Sicilian type). Am. some of the CF tests. Thanks are algOdue to the J. Trop. Med. Hyg., 25: 456-462. technicians and field workers of the IEC for their 9. Srihongse, S., and Johnson, C. M., 1974. Human invaluable support and dedication throughout the infections with Chagres virus in Panama. Am. stud{es. J. Trap. Med. Hyg., 23: 690--693. 10. Laughlin, L. W., Meegan, J. M., Strausbaugh, L., Morens, D. M., and Watten, R. H., 1979. Ep- idemic Rift Valley rever in Egypt: Observations of the spectrum of human illness. Trans. R. Soco 1. Clarke, D. H., and Casais, J., 1958. Techniques Trap. Med. Hyg., 73: 630-633. for hemagglutination and hemagglutination-in-

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