TRANsAtnoNS OF THE ROY AL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2003) 97, 60-62 Isolations of yellow fever virus from Haemagogus leucocelaenus in Rio Grande do Sul State, Brazil Pedro F. C. Vasconcelosl, Alethéa F. Sperb2, Hamilton A. O. Monteirol, Maria A. N. Torres2, Maria R. S. Sousal, Helena B. Vasconcelosl, Lúcia B. L. F. MardinP andSueli G. Rodriguesl lWHO Collaborating Center for Arbovirus Reference and Research.. Department of Arbovirus..Instituto Evandro Chagas.. Fundação Nacional de Saúde (FUNASA).. Ministério da Saúde (MS).. Belém.. PA.. Brazil; 2Secretaria de Saúde do Estado doRio Grande do Sul.. Porto Alegre.. RS.. Brazü , Abstract Following howling monkey (Alouatta caraya) deaths and yellow fever (YF) antigen detection by immunohistochemistfy in the liver sample of a dead monkey in April and May 2001 in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, epiderniological field investigations were initiated. Two strains ofYF virus were isolated in suckling mice from 23 Haemagogus (Conopostegus) leucocelaenus Dyar & Shannon mosquitoes collected from the study sites. The YF virus was isolated from this species in the 1930s in Brazil and in the 1940s in Colombia. No human cases were reported during the current epizootic outbreak. The YF virus isolation and the absence of Hg. (Haemagogus) janthinomys Dyar from the area suggest that Hg. leucocelaenus may be a secondary YF vector and play an important role in the epidemiology of this disease in the Southern Cone. Keywords: yellow rever, Haemagogus leucocelaenus, transmission, control, virus isolation, Brazil YF) in places where monkey deaths had occurred, between 09:00 and 15:00. Collections were made both at ground leveI and in the forest canopy at an elevation of appro~mately 15 m. After collection, insects were immediately frozen in liquid nitrogen for transport to the laboratory and storage at -70 oCo Specimenswere soned under a stereoscopicmicroscope on a chill table fito pools according to method of collection, locality, date of capture, and genus. The size of pools varied from 1 to 50 insects. lntroduction The yellow rever (YF) vírus is currently endemic in South America and Africa, affecting as many as 200000 persons annually (Monath, 2001). The vírus has been reported from nearly two-thirds of Brazi1ian territory; but the coastal region is generally considered free of YF, including states on the northem, south- eastemand southem coasts (Monath, 1997; Vasconce- los et al., 1999). In the past, the YF-endemic zone included Rio Grande do Sul State which is located on the Southem Cone border with Argentina and Uruguay (Taylor, 1951). The last YF cases reported in Rio Grande do Sul State occurred in 1966. Since then, no human or monkey cases have been reported (Nobre et al., 1994). In April and May 2001, several monkey deaths occurred in the Uruguay river basin in the municipalities of Garruchos and Santo Antônio das Missões and were reported to the Rio Grande do Sul State Department of Health. This paper describes the results obtained during an epidemiological study car- ried out in the area. Virus isolation and identijication Individual mosquito pools were triturated in sterile grinders containing I mL of phosphate-buffered saline solution with 0.75% bovine albumin, penicillin (100 units/mL) and streptomycin (100 ~/mL). The resul- tant suspension was centrifuged at about 1800 g for 5 mino The supernatant was withdrawn and divided into two parts: one portion was refrozen at -70 °C for reisolation attempts, the other was inoculated intracere- brally (0.02 mL) into a litter comprising six newborn albino mice. Inoculated animaIs were observed daily, and mice were collected if they showed signs of disease or died. Virus isolateswere identified by a complement fixation test using specific antiserum for YF, and by reverse transcriptase-polymerase chain reaction assays (Beaty et al., 1989; Lanciotti et aI., 1992). The mini- mum infection rate (MIR) of Haemagogus leucocelaenus Dyar & Shannonmosquitoes was calculated by dividing the total number ofYF-positive pools by the total num- ber of specimens processed(Walter et aI., 1980). Materiais and Methods Studyareas and collection methods Field studies were carried out at two localities in Rio Grande do Sul State (Figure) from 13 to 26 May 2001, following the deaths of 'bugios' monkeys (Alouatta caraya) , apparently as a consequence of a haemorrhagic rever strongly suggestive of YF. The first study site, Garruchos (55°45'W, 28°17'S), is a rural municipality with 3676 inhabitants and the second, Santo Antônio das Missões (55°21'W, 28°29'S), has 12690 inhabi- tants. Santo Antônio das Missões is 13 km from Garru- chos, and both municipalities are about 650 km inland from Porto Alegre city, the Rio Grande do Sul State capital (Figure). Climatic conditions at the two sites are similar. Mean annual temperature is 22.1 °C (range 15.5-26.1 °C), with high humidity (71.1%:f: 10.4 SD) and relatively little variation throughout the year. There are two distinct seasons,with a 4-month wet season Ganuaryto April) and an 8-month dry season(May to December). Annual rainfall averages 164.2 mm for both municipalities, with about 80% of the rain occur- ring during the wet season.Mosquitoes were captured from human bait (alI humans were vaccinated against Results In Garruchos 237 mosquitoes were captured and prepared as 31 pools for virus isolation, and in Santo Antônio das Missões 481 mosquito specimens were collected and processedas 33 pools for virus isolation (Table). The 23 (4 pools) of Hg. leucocelaenus mosqui- toes were recognized as potential secondary YF vectors and two YF strains were isolated from the Hg. leucoce- laenus collected in Santo Antônio das Missões. No viroseswere isolated from the other 62 mosquito pools. The overall MIR for Hg. leucocelaenus was 8.7%. DiscussioD Between 1998 and 2001, YF occurrence increased in Brazil, and outbreaks were reponed in both northem and south-eastem regions (Vasconcelos et ai., 2001a, 2001 b). The monitoring of YF outbreaks has increased and several epizootic areas have been recognized,some- times based on rumours of monkey deaths. Investiga- Address for correspondence: Dr Pedro F. C. Vasconcelos, WHO Collaborating Center for Arbovirus Reference and Research, Department of Arbovirus, Instituto Evandro Cha- gas/FUNASA, Av. Almirante Barroso, 491, 66090-000, Be- lém, PA, Brazil; e-mail pedrovasconcelos@iec.pa.gov.br