Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil.

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10 and 10 Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed. UNITERMS: Encephalitis viruses, physiology. Psorophora ferox. Aedes scapularis. Aedes serratus. Insect vectors, microbiology. Encephalitis, epidemic, transmission. Arbovirus infection. * Division of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Departmente of Health and Human Services, P.O. Box 2087, Fort Collins, Colorado 80522 USA. ** Department of Epidemiology, Faculdade de Saúde Pública, Universidade de São Paulo, Av. Dr. Arnaldo, 715 01255 São Paulo, SP Brazil. *** Use of trade names or commercial sources is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services. Rocio virus was responsible for several epidemics of meningoencephalitis in coastal communities in Southern S.Paulo, Brazil, during 1975 and 1976. The natural transmission cycle has not been defined, but there is strong evidence to indicate that the virus is cycled between mosquitoes and birds (Iversson, 1977, 1980; Forattini et al., 1978, 1981; Lopes et al., 1978, 1981; Mitchell et al., 1981; Mitchell & Forattini, 1984). Studies on mosquitoes captured during the epidemic yielded a single isolate of Rocio virus from a pool of Psorophora ferox (Von Humboldt) that contained engorged as well as unengorged specimens (Lopes et al., 1981). Forattini et al. (1961, 1978) reported that the predominant mosquito species in the epidemic area are Aedes serratus (Theobald), Aedes scapularis (Rondani), and Culex (Melanoconion) species. Mitchell & Forattini (1984) demonstrated that Ae. scapularis from the epidemic zone in Brazil is an efficient vector of Rocio virus under experimental conditions. We report here on experiments with Ps. ferox and Ae. serratus and, for comparison, include additional data on Ae. scapularis. MATERIALS AND METHODS Virus. The Rocio virus strain used (SpH34675) was isolated at autopsy in 1975 from human brain tissue. Viral stocks were 10% suspensions of infected suckling mouse brain from the third passage. Mosquitoes. Moesquitoes were collected aperiodically from December 1981 through March 1985 in "Pariquera-Açu" Township, S.Paulo State, Brazil, and brought to the laboratory at the University of S.Paulo. Females were given a blood meal and allowed to oviposit on filter paper. Eggs were conditioned for 10 days at 28°C in a humid atmosphere, then packaged in plastic bags and sent to the Division of Vector-Borne Viral Diseases, Centers for Disease Control, in Fort Collins, Colorado, via airmail. Usually, eggs were flooded in deionized water on the day received. Occasionally, a vacuum pump or nutrient broth was utilized in an attempt to improve hatching success. Tetramin*** fish food and commercial rabbit chow were given ad libitum until pupation occurred. Adult females were given 5% sugar water from the time of emergence until 24 h before a blood meal was offered. Only F1-generation females were used experimentally, since an objective was to measure the susceptibility of field populations and not that of laboratory-adapted colonies. Experimental procedure. The experimental design is essentially the same as that described in previous vector competence studies involving Rocio virus (Mitchell et al., 1981; Mitchell & Forattini, 1984). Chicks less than 48 h old were infected with Rocio virus by subcutaneous inoculation of ca. 10,000 Vero cell plaque-forming units (PFU). Mosquitoes 3 to 9 days of age were allowed to feed overnight on viremic chicks 54 to 72 h postinoculation. Chicks were restrained on top of 1/2-pint cartons that were covered with fine-mesh nylon and that contained, the mosquitoes. When feeding two species simultaneously on the same chick, the chick was sandwiched between two cartons that were taped together. Postexposure chick bloods were drawn by jugular venipuncture and frozen at -70°C until tested for virus. Engorged mosquitoes were segregated, provided oviposition dishes, and incubated at 26.7 ± 0.5°C, 75% to 80% RH, and a photoperiod of 16 h full light and 8 h of darkness. Some mosquitoes were given an opportunity to refeed individually on 1to 2-day-old chiks following appropriate periods of incubation. The mosquitoes were frozen, and chicks that were bitten were banded and bled about 60 h later for virus assay. In one series of experiments, the in vitro feeding technique described by Aitken(l977) was used to demonstrate virus transmission by mosquitoes. Briefly, glass capillary tubing drawn to a fine point in the flame of an alcohol lamp was marked with a rubber stamp at 1-mm increments. Each increment, corresponded to an internal volume of 0.17ml. Each capillary tube was loaded with 5 ml of fetal calf serum (FCS) at pH 7.2, and the proboscis of a test mosquito was inserted following removal of the mosquito's wings. The capillary tube with dangling mosquito was transferred to a styro foam rack, and the mosquito was allowed to feed for approximately 15 min. The amount of feeding suspension ingested by each mosquito was recorded. The remainder of the suspension was expressed into a microscope slide, loaded into another calibrated capillary tube, and infected parenterally into from one to four colony Cx. pipiens from Dayton, Ohio. The inoculation apparatus used was that described by Rosen & Gubler (1974). These inoculated mosquitoes were given 5% sugar water and incubated at 26.7°C for 7 days. At that time, they were frozen at -70°C until processed and tested for virus as described below. Mosquitoes used in the in vitro feeding trials were frozen immediately after having their proboscis removed from the capillary. Virus content of each of these donor mosquitoes was determined by titration in Vero cell culture, and this information was collated with the amount of feeding suspension ingested. General procedures used for processing mosquitoes for virus isolation tests have been described (Sudia & Chamberlain, 1967). Mosquitoes were disrupted by sonic energy in 1 ml of BA-1 diluent (0.2 M Tris, pH 8.0, 0.15 M NaCl, 1% BSA, 10 mg/litre phenol red, 50 g/ml Gentamicin, and 1 g/ml Fungizone). Suspensions were centrifuged at 2000 rpm for 20 min. The supernatant was frozen at -70°C until tested. Blood samples (0.1 ml) from chicks were taken by jugular venipuncture diluted in BA-1, centrifuged at 1500 rpm for 15 min, dispensed into screw-cap vials, and stored at -70°C. Specimens were screened for virus or were titrated as appropriate. Viral assays were done by inoculating Vero cell cultures and counting plaques. Briefly, tenfold dilutions were made in BA-1, and samples (0.1 ml) were inoculated into Vero cell cultures in six-well plates, adsorbed for 1 h at 37°C, and overlaid with 1% Noble agar in M-199 supplemented with 2% PCS, 2.0 g/litre of NaHC03, 150 g/ml of DEAE-dextran, and 1:40,000 neutral red. Cell cultures were then examined for 10 days for characteristic plaques. The virus infection rate in mosquitoes, expressed as a percentage, is the proportion of mosquitoes tested that contained virus. The ID50 value was estimated using probit regression. The virus transmission rate, also expressed as a percentage, is the proportion of infected mosquitoes that transmitted virus upon refeeding after a suitable extrinsic incubation period. Differences in infection rates between species fed simultaneously on the same viremic chick were tested for significance by Fisher's Exact Test (Snedecor & Cochran, 1967).