An overview of Arbovirology in Brazil and neighbouring countries. Edited by: Amélia P. A. Travassos da Rosa Pedro F. C. Vasconcelos Jorge F. S. Travassos da Rosa BELÉM INSTITUTO EVANDRO CHAGAS 1998 Characterization of two new phleboviruses associated with human illness from the Amazon Region of Brazil. Sueli Guerreiro RODRIGUESI Arnélia P.A. TRAVASSOS DA ROSAI Pedro F.C. V ASCONCELOSI Elizabeth S. TRAVASSOS DA ROSAI Robert B. TESH2 Jorge F.S. TRAVASSOS DA ROSAI I. Centro Colaborador de Investigação e Adestramento em Arboviroses da Organização Mundial da Saúde, Serviço de Arbovirus, Instituto Evandro Chagas, Fundação Nacional de Saúde, Ministério da Saúde. Av. Almirante Barroso, 492, 66090-000, Cx. Postal 1128, Belém, Pará, Brazil. 2. University ofTexas, Medical Branch at Galveston. Center for Tropical Diseases, Department ofPathology, 301 University Boulevard, Galveston, Texas, 77555-0609, USA. 100 INTRODUCTION The phleboviruses are agents which have shown varying degreesof cross-reactivity by standard serological tests. On the basis of their physicochemical properties and morphogenesis, they are included in the family Bunyaviridae, genus Phlebovirus (Bishop & Shope, 1979). These viroses in general have a rather limited host range (Tesh, 1988). At present, there are 38 distinct virus serotypes registered at the International Catalogue of Arlioviruses including certain other viroses of vertebrates (Karabatsos, 1985). Eight phleboviruses (Alenquer, Candiru, Chagres, Naples, Ponta Toro, Rift ValIey Fever, Sicilian and Toscana)have been isolated from humanoWith the exception ofRift ValIey Fever which have causedhemorrhagic rever and Toscana which is associatedwith central nervous system disease, the human ilIness produced by the remaining six agents is identical and is characterized by an acute self-limited flu-like ilIness oftwo to tive days duration (Tesh, 1988; Nicoletti ~., 1991) In the Brazilian Amazon, 21 (nine unregistered)phleboviruses have beenisolated (Table 1). Ofthese, four (Alenquer, Candiru, Morumbi and Serra Norte) were obtained from the blood offebrile patients. These four agents pIos ltaituba virus forro the Candiru Complex within the Phlebotomus rever serogroup. AlI of these viroses are indigenous to the Amazon Basin and have not been isolated outside of this region. This present paper describes the isolation, antigenic relationships and clinical picture associated with disease in humans of Morumby and Serra Norte viroses, two new members of the Candiru complex, which were obtained from febrile humans. Table 1. Phlebovirusesisolated in the Brazilian Amazonaccording with the source,material, place and date of isolation. VIRUS DATE MATERIAL SOURCE PLACE ** ALENQUER 05-31-76 Blood Human Alenquer AMBÉ 01-14-82 Arthropod Phlebotominaesp Altamira ANHANGA 10-01-62 Viscera Choloepusbrasiliensis Castanhal BELTERRA 09-22-78 Viscera Proechimyslongicaudatus Belterra BUJARU 10-26-62 Blood Proechimysguyannensis Belém CANDIRU 09-27-60 Blood Human Ipixuna ICOARACI 10-16-60 Viscera Nectomyssquamipes Belém ITAITUBA 12-08-71 Blood Didelphis marsupialis Itaituba ITAPORANGA* 11-10-63 Viscera Sentinelmouse Belém JACUNDÁ 10-31-84 Viscera Myoproctaagouchi Tucuruí JOA 03-29-79 Arthropod Lutzomyiasp Altamira MORUMBI 04-12-88 Blood Human Tucuruí MUNGUBA 09-20-80 Arthropod Lutzomyiaumbratilis Monte Dourado O RIXIMIN Á 07-03-80 Arthropod Lutzomyia sp Oriximiná PAcuí 02-10-61 Blood Oryzomysgoeldi Ipixuna RONDÔNIA 12-30-88 Arthropod Phlebotominaesp Samuel SERRA NORTE 03-01-91 Blood Human Carajás TAPARÁ 62-04-83 Arthropod Phlebotominaesp Altamira TURUNA 07-26-78 Arthropod Lutzomyia sp Oriximiná URIURAN A 12-16-85 Arthropod Phlebotominaesp Tucuruí URUCURÍ 04-19-66 Blood Proechimysguyannensis Belém * Prototype was obtained in São Paulo by Ewald Trapp at Instituto Biológico de São Paulo ** AlI viruses were isolated in Pará State with the exception ofRondônia vírus which was isolated in Samuel, Rondônia State. 101 Figure 1 -Map of Pará State, showing,the places of isolation of the phlebovirusesMorumbi (Tucuruí)and SerraNorte (Carajás). MATERIALS AND METHODS Virus isolation Blood sampleswere collected by venopuncture.Once obtained, aliquots of SerUD1were immediatelypreserved at -70°C until used in laboratory. Samples were diluted in PBS solution with 0.75% of bovine albumin, containing antibiotics and iJ1oculatedintracerebrally intoa group of six suckling Swiss albino mice (Shope & Sather, 1979). Virus identification Viroses were identified by compIement fixation (CF) test according to a microtechnique modified (FuIton & DumbeII, 1949), using two fuII units of guinea pig complementand mouse neutralization (NT) in suckling mice, using a final dilution of 1 :8. Serom -vírus mixtures were incubated for one hour at 37°C and then inoculated intracerebralIy, and for pIaque reduction neutralization test (PRNT) (Casais, 1963; Shope & Sather, 1979). Antigens Antigenswere preparedfrom infectednewborn mouse brains by the sucrose-acetonemethod (Clarke & CasaIs,1958). Later, hemagglutinationactivity was testedusing the extractedantigens. Virus identification Viroses were identified by compIement fixation (CF) test according to a microtechnique modified (FuIton & DumbeII, 1949), using two fuII units of guinea pig complementand mouse neutralization (NT) in suckling mice, using a final dilution of 1 :8. Serom -vírus mixtures were incubated for one hour at 37°C and then inoculated intracerebralIy, and for pIaque reduction neutralization test (PRNT) (Casais, 1963; Shope & Sather, 1979). 102 RESULTS Antigenic relationships By cross CF tests both Morumbi and Serra Norte viroses are closely related to ltaituba virus, another memberofthe Candiru complex ofthe Phlebotomusrever serogroup. However, the results of cross neutralization (Table 2) and PRNT (Table 3) clearly separate eachvirus. Table 2. Serological results oí cross NT tests with phleboviruses oí Candiru complex isolated in Brazil, including Morumbi and Serra Norte viroses. ANTISERA103 c:> CANDIRU ITAITUBA JACUNOA MORUMBI SERRA NORTE VIRUS fJ CANDIRU 103* 2.5 1.12.1 0.8 1.3 ITAITUBA 4.0 103 1.22.8 2.72.8 JACUNDÁ 10382 1.6 3.1 103 103 MORUMBI 103 1.2 0.2 2.6 1.2 SERRA NORTE 3 3.3 2.3 1.3 4.0 * Homologoustiters Neither Morumbi or SerraNorte virus had hemagglutinationactivity with gooseerythrocytes. Mosquito celI culture AlI viroses of Candiru complexwere cultived in C6/36 cells. Characteristiccytopathogenic effect was observedthree days after inoculation. Clinical symptoms Both individuaIs had an abrupt onset with mild to high rever, chills, headache, myalgia, photophobia, retro-bulbar paio and low back paio. The symptoms remained for four days. The convalescent period was uneventful. Arthralgias, weaknessand dizziness were algo observed in Morumbi disease. Morumbi virus was obtained from a 52 year old male, in Novo Repartimento county, Tucurui Town, Pará State. Serra Norte vírus was isolated from a 42 year old male laboratory worker. The látter individual was a part ofthe leishmaniasis program ofInsituto Evandro Chagas and was collecting sand flies in Carajás area, Pará State (Figure 1). DISCUSSION Based on CF results, the existence ofseven antigenic complexeshas beenproposed within the Phlebotomus rever serogroup (Travassos da Rosa ~., 1982; Tesh ~., 1982). With the exception ofRift Valley rever virus (RVF) and Toscana (TOS), alI phleboviruses isolated from humans have been associated with systemic febrile ilIness that cover a wide clinical pattem ranging from simple rever to a characteristic déngue-like syndrome. RVF virus in Africa, Canalgo cause hemorrhagic rever and meningoencephalitis with a lethality cate ranging between1 % to 5% (Brés, 1988). TOS virus have been associated with CNS di$ease(meningitis and meningoencephalitis)in ltaly and probably other countries in Mediterran. Recently, Nicoletti ~.t.g.l(1991) published convincent data suggestingTOS virus as the causative agent of 155 casesofmeningitis and meningoencephalitisin the Tuscany and Marche regions ofItaly. These data, show the importance ofthe phleboviruses as agents ofhuman diseases. The diagnosis of Phiebovirus infection is difficult to make, and unless special attention is given to such cases, they are readly confused with malaria, influenza andother respiratory viTal diseasesor other arboviral infection. Phleboviruses are generalIy transmitted by bite of sand flies. Humans are infected when they intrude into the insect's ecological niche. In the New World, this tangencial mode of infection results in sporadic and lirnited number of cases, usualIy in people living near or in forests. The frequency of transrnission of these viroses to mau and domestic animais dependsofthe vector population density,seasonally, temperature, hurnidity, etc. The phleboviruses probably have a limited distribution within their ecologic foci. 103 3.7 Table 3. Results of plaque reductionneutralization tests with SerraNorte and Morumbi viruses, using 46 differentphlebovirus antibodies. Antibody Homologoustiters Virus Morumbi Serra Norte Aguacate 1,280 o o Alenquer 80 ~40 o Ambe 160 10 o Anhanga 320 10 o Arbia 160 ~40 o Arboledas 1,280 O o ATn1ero 40 O o Arumowot 5,120 O o Belterra 320 ~40 o Buenaventura 40 ~40 o Bujaru 1,280 20 o Cacao 80 O o Caimito 40 O o Candiru 1,280 ~40 40 Chagres 2,560 ~40 O Chilibre 1,280 O O Corfu 160 O O Durania 40 O O Frijoles 5,120 O O GabekForest 320 O O Gordil 10,240 O O Icoaraci 10,240 ~40 O Itaituba 5,120 ~40 340 Itaporanga 160 O O Ixcanal 320 O O Joa 10,240 ~40 O Karimabad 320 10 O Mariquita 320 O O Munguba 5,120 O O NaplesNique