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Characterization of Eight New Phlebotomus Fever Serogroup Arboviruses (Buny Aviridae: Phlebovirus) from the Amazon Region of Brazil*

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Characterization of Eight New Phlebotomus Fever Serogroup Arboviruses (Buny Aviridae: Phlebovirus) from the Amazon Region of Brazil* 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 ROBERT B. 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.
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