Characterization of Five New Phleboviruses Recently Isolated from Sand Flies in Tropical America

CHARACTERIZATION OF FIVE NEW PHLEBOVIRUSES RECENTLY ISOLATED FROM SAND FLIES IN TROPICAL AMERICA

ROBERT B. TESH, JORGE BOSHELL, DAVID G. YOUNG, ALBERTO MORALES, CRISTINA FERRO DE CARRASQUILLA, AUGUSTO CORREDOR, GOVIND B. MODI, AMÉLIA P. A. TRAVASSOS DA ROSA, ROBERT G. McLEAN, CRISTINA DE RODRIGUEZ, AND MARTA O. GAITAN Yale University School of Medicine, New Haven, Connecticut; Instituto Nacional de Salud, Ministério de Salud, Bogotá, Colômbia; University of Florida, Gainesville, Florida: Instituto Evandro Chagas, Para, Brazil; and Centers for Disease Control, Fort CoUins, Colorado

Abstract. Five new phlebotomus fever virus serotypes (Bunyaviridae: Phlebovirus) are described. These viruses, designated Ambe, Ixcanal, Mariquita, Armero, and Durania, were isolated from sand flies (Diptera: Psychodidae) collected in Brazil, Colômbia, and Guatemala. Two of the agents were recovered from pools of male sand flies. The new viruses are antigenically related to other members of the phlebotomus fever serogroup by immunofluorescence, but are distinct from the other 39 members of this serogroup by plaque reduction neutralization test.

A total of 39 distinct virus serotypes have been ascitic fluids (strain identifications are given in described in the phlebotomus fever serogroup parentheses): Aguacate (VP-175 A), Alenquer (Be (Bunyaviridae: Phlebovirus).^- These show H 301101), Ambe (Be Ar 407981), Anhangá (Be varying degrees of antigenic relatedness by dif- An 46852), Arbia (ISS Phl 18), Arboledas (Co ferent serologic tests.'-" For example, in Ar 170152), Armero (Co Ar 171096), Arumowot hemagglutination inhibition (Hl) and immuno• (Ar 1284-64), Belterra (Be An 356637), Bue- fluorescence (IFA) tests, many of the phlebovirus naventura (Co Ar 3319), Bujaru (Be An 47693), serotypes are broadly cross-reactive,' thus these Cacao (VP-437R), Caimito (VP-488A), Candiru techniques are useful in establishing serogroup (Be H 22511), Chagres (JW-10), Chilibre (VP- relationships. The complement fixation (CF) test 118D), Corfu (Pa Ar 814), Durania (Co Ar is more specific;'-^ by this method most of the 171162), Frijoles (VP-161 A), Gabek Forest (Sud serotypes can be difFerentiated, although a few An 754-61), Gordil (DakAn BR 496d), Icoaraci antigenic complexes which contain 2 or more (BeAn 24262), Itaituba (BeAn 213452), Itapo- serotypes indistinguishable by CF test exist with- ranga (original), Ixcanal (CA Ar 170897), Joa in the phlebotomus fever serogroup (i.e., Naples, (BeAr 371637), Karimabad 1-58), Mariquita A Candiru, and Salehabad complexes). The plaque (prototype), Munguba (BeAr 389707), Nique reduction neutralization test (PRNT) is the most (Nique-9c), Odrenisrou (DakAr Al 131), Orixi- specific serologic method for identifying mem• mina (BeAr 385309), Pacui (BeAn 27236), Punta bers of this serogroup;^-" consequently it is the Toro (D-40210A), Rift Valley fever (Entebbe), technique which is presently used to define virus Rio Grande (TBM4-719), Saint Floris (Dak An serotypes within the genus Phlebovirus. BR 512d), Salehabad (1-81), sand fly fever-Na• Here we describe 5 new phlebotomus fever ples (Sabin), Sand fly fever—Sicilian (Sabin), serotypes isolated from sand flies (Diptera: Psy• Teheran (1-47), Toscana (ISS Phl.3), Turuna chodidae) collected in Brazil, Colômbia, and (BeAr 352492), and Urucuri (BeAn 100049). Guatemala. Stocks of each virus were prepared from infected mouse brain or infected Vero cells. MATERIALS AND METHODS Viruses Immune reagents The following phleboviruses were used in neu• The immune serum against Rift Valley fever tralization tests and to prepare immune sera or virus was prepared in a sheep inoculated with 529 530 TESH AND OTHERS the Entebbe virus strain. This serum was pro- rania, Colômbia. Many of the subjects in Dur- vided by C. J. Peters of the U.S. Army Institute ania were leishmaniasis cases; most of the sub• of Infectious Diseases, Frederick, MD. Specific jects in both communities were adolescents and hyperimmune ascitic fluids (MIAF) against each adults. of the other 43 viruses were prepared in mice, Wild animais were trapped live on the periph- using either infected newborn mouse brain or ery of Durania in a secondary tropical forest. infected Vero cells as the immunizing antigen. This was located several km from the sand fly The immunization Schedule for the mice con- collection site. Blood was obtained from anes- sisted of 4 ip injections given weekly. Immuniz• thetized animais by cardiac puncture. ing antigens were mixed with 1 ml of Freund's complete adjuvam just prior to inoculation. Sar• RESULTS coma 180 cells were also given ip with the final injection to induce ascites formation. Ambe virus

Virus isolation techniques Ambe virus, prototype strain Be Ar 407981, Ambe virus was originally isolated in newborn was isolated at the lEC from a pool of 150 male mice at the Instituto Evandro Chagas (lEC) in sand flies which were coUected on 14 January Belém. The other 4 viruses were isolated in Vero 1982 from tree trunks in a forested area near cell cultures at the Instituto Nacional de Salud Altamira, Para State, Brazil. Ambe virus was (INS) in Bogotá or at the Yale Arbovirus Re• initially isolated in newborn mice; the average search Unit (YARU) in New Haven, CT. The survival time of baby mice after ic inoculation techniques used for processing field coUected sand of the virus is 6 days. In CF tests done in Belém, flies for virus isolation have been described.- Ambe antigen reacted weakly with Icoaraci and Belterra MIAF. By IFAT, it reacted with the Virus identification phlebovirus fever grouping reagent and with 2 of the specific phlebovirus MIAF. However, in Characterization of the isolates was done at PRNT, Ambe virus was not neutralized by any YARU. The viruses were initially screened by of the 43 heterologous MIAF (Table 1). There- indirect fluorescent antibody technique (IFAT)^ fore, it appears to be a new phlebovirus. against 6 different immune reagents: 3 broadly reacting polyclonal (grouping) antibodies which reacted with viruses in the vesicular stomatitis, Ixcanal virus phlebotomus fever, and Changuinola sero- groups, and 3 specific mouse immune ascitic This agent, prototype strain CA Ar 170897, fluids made against Arboledas, Chagres, and was also isolated from a pool of male sand flies Icoaraci viruses. Infected Vero cells, grown on (Lutzomyia sp.) at YARU. The insects were col- Lab Tech chamber slides (Miles Laboratories), lected on 22 and 23 November 1982 during served as antigens for the IFAT. Immune re• leishmaniasis studies in Aldeã Ixcanal and Aldeã agents were used at a 1:10 dilution. Puerta Progreso, Guatemala. The 2 aldeãs are Viruses which reacted in the IFAT with the located in the riparian habitat of the Cato River phlebotomus fever grouping reagent or with 1 or Valley on the south slope of the Sierra de las more of the specific phlebovirus antibodies were Minas Mountains at ~400 m elevation. Ixcanal then screened by PRNT against the 44 specific virus was originally isolated in Vero cell cultures; phlebovirus MIAF noted above. The PRNT was it did not produce disease in baby mice despite performed in microplate cultures of Vero cells 5 intracerebral (ic) blind passages. By IFAT test, using a fixed virus inoculum and varying dilu- Ixcanal virus is a member of the phlebotomus tions of antibody. The technique has been de- fever serogroup; by PRNT it was found to be a scribed previously.^ The highest antibody dilu• new serotype (Table 1). tion producing >90% plaque reduction was recorded as the endpoint. Mariquita virus Human and animal sera This virus, prototype strain Mariquita A, was Sera were coUected from a sample of people isolated at the INS from a pool of female sand visiting local hospitais in Mariquita and Du- flies coUected in January 1985 from human bait FIVE NEW PHLEBOVIRUSES FROM CENTRAL AMERICA 53 1

TABLE 1 Results of plaque reduction neutralization tests with the 5 new phleboviruses

Virus (Homologous Antibodv titer)* Ambc Ixcanal Mariquita Armero Durania Aguacate (1,280) 0* 0 0 0 0 Alenquer (80) 0 0 0 0 0 Ambe — 760 0 0 0 0 Anhangá (320) 0 0 0 0 0 Arbia (160). 0 0 0 0 0 Arboledas (1,280) 0 0 0 0 0 Armero _ 0 0 0 40 0 Arumowot (5,120) 0 0 0 0 0 Belterra (320) 0 0 0 0 0 Buenaventura (40) 0 0 0 0 0 Bujaru (1,280) 0 0 0 0 0 Cacao (80) 0 0 0 0 0 Caimito (40) 0 0 0 0 0 Candiru (1,280) 0 0 0 0 0 Chagres (2,560) 0 0 0 0 0 Chilibre (1,280) 0 0 0 0 0 Corfu (160) 0 0 0 0 0 Durania _ 0 0 0 0 40 Frijoles (5,120) 0 0 0 0 0 Gabek Forest (320) 0 0 0 0 0 Gordil (10,240) 0 0 0 0 0 Icoaraci (10,240) 0 0 0 0 0 Itaituba (5,120) 0 0 0 0 0 Itaporanga (160) 0 0 0 0 0 Ixcanal 0 320 0 0 0 Joa (10,240_ ) 0 0 0 0 0 Karimabad (320) 0 0 0 0 0 Mariquita ( ) 0 0 320 0 0 Munguba (5,120) 0 0 0 0 0 Naples (320) 0 0 0 0 0 Nique (80) 0 0 0 0 0 Odrenisrou (640) 0 0 0 0 0 Oriximina (160) 0 0 0 0 0 Pacui (2,560) 0 0 0 0 0 Punta Toro (1,280) 0 0 0 0 0 Rift Valley fever (320) 0 0 0 0 0 Rio Grande (320) 0 0 0 0 0 Saint Floris (5,120) 0 0 0 0 0 Salehabad (320) 0 0 0 0 0 Sicilian (5,120) 0 0 0 0 0 Teheran (80) 0 0 0 0 0 Toscana (5,120) 0 0 0 0 0 Turuna (1,280) 0 0 0 0 0 Urucuri (640) 0 0 0 0 0

* Reciproca] of híghest antíbody dilution producing >90% plaque reduction; O = 1:10. in an área of secondary moist tropical forest in Armero virus the municipality of Mariquita, Department of Tolima, Colômbia. Mariquita virus was initially This agent, prototype strain designated Co Ar recovered in Vero cell cultures. On primary ic 171096, was isolated at YARU from a pool of inoculation into baby mice, it produced illness 100 female Lutzomyia sp. captured from human on day 7; on subsequent passage, it killed new- bait on 21 January and 6 February 1986 at the bom mice in 3-4 days. By IFA, Mariquita virus same locality where Mariquita virus was ob- is a member of the phlebotomus fever serogroup. tained. Armero virus was isolated initially in Vero By PRNT it was distinct from the other 43 known cell cultures; on primary inoculation into new- phleboviruses (Table 1). born mice, it produced illness on day 9. By IFA, 532 TESH AND OTHERS

Armero virus is a member of the phlebotomus reassortment and new serotypes are continuously fever serogroup; by PRNT it appears to be a new evolving. Phieboviruses also have segmented ge- serotype (Table 1). nomes,* and it is possible that they behave sim- ilarly. Durania virus To date, the method used to establish new viruses within the phlebotomus fever serogroup This virus, prototype strain Co Ar 171162, was has been the PRNT. Of the 5 commonly used also isolated at YARU from a pool of 100 female serologic techniques (CF, Hl, IFAT, PRNT and sand flies captured from human bait on 13 July ELISA), the PRNT is the most specific.'-'^ Be• 1986. The collection site was a coffee plamation cause of varying degrees of cross-reactivity among located about 8 km from Durania, Department the phieboviruses, it is not possible to differen- of North Santander, Colômbia. Durania virus tiate all of the serotypes by CF, Hl, IFAT or was isolated in Vero cell cultures; on its first ELISA. The PRNT is not entirely satisfactory. passage in baby mice, it produced hind limb pa- During the past 20 years, we have isolated a num• ralysis and death 12-13 days after ic inoculation. ber of phieboviruses from sand flies in Brazil, Durania virus is a member of the phlebotomus Colômbia, and Panamá which are probably new fever group by IFA; but by PRNT, it is distinct serotypes but which cannot be fully characterized from the other 43 known serotypes (Table 1). because they do not produce readable plaques in In an attempt to determine if any of the new tissue culture under agar. These agents produce viruses infect humans, 109 sera from hospital cytopathic effect in Vero cells and in some cases patients in Durania were screened at a 1:10 di- kill newborn mice, but they are otherwise diflS- lution by PRNT against Durania virus. Likewise, cult to study. Given the number and diversity sera from 108 hospital patients in Mariquita were among the phieboviruses, other techniques must screened by the same technique for neutralizing be developed to study and characterize new iso- antibodies to Armero virus. None of the human lates. It seems probable that many yet unrecog- sera from these communities had detectable neu• nized serotypes exist in nature, since sand flies tralizing antibodies against either virus. as a group have been neglected. Most workers have concentrated on mosquitoes and ticks, which are generally easier to collect and identify. DISCUSSION Two of the new phlebotomus fever serotypes The addition of these viruses to the phlebot• (Ambe and Ixcanal) were isolated from pools of omus fever serogroup brings the total number of male sand flies. This is not a new finding." How- recognized serotypes to 44. Most of the viruses ever, it does emphasize the apparent importance in this serogroup have been associated with phle- of transovarial transmission in the maintenance botomine sand flies, and the majority (69%) have of this group of viruses in nature. It also dem- been isolated in tropical America.' The pre- onstrates the utility of male sand flies in arbo- ponderance of phlebovirus serotypes from the virus research. The insects which yielded these New World may be a reflection of the greater new viruses were collected during field studies species diversity of sand flies in the Américas. on the epidemiology of leishmaniasis. In this dis- Approximately 360 different species oí Lutzom- ease, only females are of interest, since males do yia are currently recognized from the New World, not bite. In leishmaniasis field studies, female while ~ 119 species and subspecies of Phlebot• sand flies are usually identifled and examined for omus have been reported from the Old World." the presence of protozoa. The males are then It is possible that the phieboviruses have fol- discarded or are used for taxonomic identifica- lowed the same pattem of evolutionary diver- tion. In our study, male sand flies were saved, gence as their principal vectors. In this regard. it frozen, and subsequently processed for virus iso- is interesting to note that viruses in the Chan- lation. Two of the male pools yielded new phie• guinola serogroup (Reoviridae: Orbivirus) also boviruses. In addition to phieboviruses, some of have tremendous serotype diversity.' These lat- the male sand flies also yielded a new virus (Ca• ter agents are also transmitted by phlebotomine rajás) belonging to the vesicular stomatitis sero• sand flies in the Neotropics. It has been postu- group (Rhabdoviridae: Vesiculovirus)." lated that, because of their segmented genome, One of the deficiencies of this study was our Changuinola viruses frequently undergo genetic failure to specifically identify the sand flies that FIVE NEW PHLEBOVIRUSES FROM CENTRAL AMERICA 533 were processed in virus isolation attempts. This ical Medicine and Hygiene. Address correspon- was due to the large numbers coUected and the dence to N. Karabatsos, Centers for Disease Control, Fort Collins, CO 80522. time required to properly examine and identify 2. Tesh RB, Boshell J, Young DG, Morales A, Cor• individuai specimens.'" Consequently, we elect- redor A. Modi GB, Ferro de Carrasquilla C, de ed to sort the sand flies by sex and then process Rodriguez C, Gaitan MO, 1986. Biology of Ar- them in poois of 50-200 insects. boledos virus, a new phlebotomus fever serogroup virus (Bunyaviridae; Phlebovirus) iso- Acknowledgments: The authors wish to acknowledge lated from sand flies in Colômbia. Am J Trop the fine technical assistance of Hector Anaya. Hilda MedHygSS: 1310-1316. UI:87073956 Guzman, Maria Teresa Palau, Enrique Martinez, and 3. Travassos da Rosa AP. Tesh RB, Pinheiro FP, Bruce Alexander. This report is No. 9067 of the Florida Travassos da Rosa JF, Peterson NE, 1983. Agricultural Experiment Station journal series. Characterization of eight new phlebotomus fe• ver serogroup arboviruses (Bunyaviridae: Phle• Financial support: The Colombian Ministry of Health; bovirus) from the Amazon region of Brazil. Am the Brazilian Ministry of the Interior; and the National J Trop Med Hyg 32: 1 164-1171. UI;84021549 Institutes of Heahh of the United States, grants AI 4. Tesh RB, Peters CJ, Meegan JM, 1982. Studies 10984 and AI 20108. on the antigenic relationship among phleboviruses. Am J Trop Med Hvg 31: 149-155. UI: Authors" addresses; Robert B. Tesh and Govind B. 82132984 Modi. Yale Arbovirus Research Unit, Department of 5. Riggs JL. 1979. Immunofluorescent staining. Epidemiology and Public Health, Yale University Lennette EH, Schmidt NJ, eds. Diagnostic pro- School of Medicine, P.O. Box 3333. New Haven, CT cedures for virai, rickettsial and chlamydial in- 06510. Jorge Boshell. Alberto Morales, Cristina Ferro fections. Sth ed. Washington. DC: American de Carrasquilla. Augusto Corredor, Cristina de Rodri• Public Health Association, 141-151. Ul:8004375 guez, and Marta O. Gaitan, Instituto Nacional de Sa- 6. Tesh RB, 1988. The genus Phlebovirus and its lud. Ministério de Salud. A.A. 80080. Bogotá. Colôm• vectors. Ar^n Rev Entomo 133: 169-181. UI: bia. David G. Young. Department of Entomology and 88132628 Nematology, University of Florida. Gainesville. FL 7. Travassos da Rosa AP. Tesh RB, Pinheiro FP, 32611. Amélia P. A. Travassos da Rosa, Instituto Travassos da Rosa JFS, Peralta PH. Knudson Evandro Chagas. Fundação Serviços de Saúde Publica, DL, 1984. Characterization of the Changuinola CP. 621, 66.000 Belém, Para, Brazil. Robert C. serogroup viruses (Reoviridae: Orbivirus). In- McLean. Division of Vector-Borne Virai Diseases, tervirology 21: 38-49. UI:84134950 Centers for Infectious Diseases, Centers for Disease 8. 1982. Classification and nomenclature of viruses. Control. P.O. Box 2087. Fort Collins. CO 80522. Fourth report of the International Committee on Taxonomv of Viruses. Inlervirologv 17: I- Reprint requests; Robert B. Tesh. Yale Arbovirus Re• 199. Ul:83006386 search Unit, Department of Epidemiology & Public 9. Travassos da Rosa AP, Tesh RB, Travassos da Health, Yale University School of Medicine, P.O. Box Rosa JF. Herve JP, Main AJ Jr, 1984. Carajás 3333, New Haven, cfo6510. and Marabá viruses. two new vesiculoviruses isolated from phlebotomine sand flies in Brazil. AmJTropMedHyg33:999-\00(>.\J\:%50204(,0 REFERENCES 10. Young DG, 1979. A review of the bloodsucking psychodid flies of Colômbia (Diptera: Phlebot• 1. American Committee on Arthropod-Borne Virus- omine and Sycoracinae). Technical Bulletin 806. es, 1985. Internmional catalogue of arboviruses Gainesville: University of Florida Institute of includingcertain other viruses of vertebrates. 3rd Food and Agricultural Sciences. ed. Karabatsos N, ed. American Society of Trop•