Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A arthropod-borne viruses 163 Aura and Una, two new group A arthropod-borne viruses* Causey, O. R. Casals, J. Shope, R. E. Udomsakdi, S. Since 1954 the Belém Virus Laboratory has been conducting a surveillance of arthropod-borne viral activity in forested areas near Belém in the Amazon valley. A description of the study area and the results obtained in the first four years have been reported in an earlier paper1. The present paper reports the isolation from mosquitoes of two new viruses, Aura (AR 10315) and Una (AR 13136), their identification as members of Casals’ group A and the available information on their epidemiology. These two viruses are named for small rivers flowing through forested areas near Belém where the original isolations were made. MATERIALS AND METHODS The methods used in processing mosquitoes for possible virus isolation have been described previously1. Initial complement-fixation (CF) and hemagglutination- inhibition (HI) testing of the two prototype isolates, AR 10315 (Aura) and AR 13136 (Una), was done in the Belém Laboratory. Immune sera were prepared in adult mice given multiple injections of live virus intraperitoneally (i.p.). CF tests were done according to a micro- technique modified from Fulton and Dumbell2, immune sera and 10% suspensions of infected mouse brain or sucrose-acetone extracted * Publicado originalmente em American Journal of Tropical Medicine and Hygiene, Baltimore, v. 12, n. 5, p. 777 - 781, Sept. 1963. 164 Memórias do Instituto Evandro Chagas: Arbovírus infected mouse brain being reacted in grid fashion. HI tests were performed according to methods described by Clarke and Casals3. Hemagglutinating (HA) antigens were prepared from infected suckling mouse brain by the sucrose-acetone technique; antigens from Mayaro virus and the Una prototype strain were treated with protamine sulfate to broaden their pH range of activity. HI tests were done at pH 6.2, 37°C. A hemagglutinin was also demonstrated in Una-infected suckling mouse serum treated with acetone, but this was not used in the tests reported here. Some extracted brain preparations of Una virus were found to be unstable on storage, even though frozen and dried. In additional HI and CF studies carried out with the prototype strains in the Rockefeller Foundation Virus Laboratories, New York, HA antigens were derived from Aura-infected suckling mouse brain both by a simple alkaline-aqueous method and by the acetone- ether method; the latter antigen had a titer of 1:12,800 in 0.4ml and a pH range of activity between 5.7 and 6.4. Una-infected suckling mouse brain yielded an antigen with greater difficulty and required use of the sucrose-acetone method followed by precipitation with protamine sulfate; the titer of this antigen was 1:160 in 0.4ml, with a pH range of activity between 6.0 and 6.3. Whenever possible, the HI tests were done with both single-injection and multiple-injection sera prepared in adult mice inoculated i.p. and guinea pigs inoculated intracerebrally (i.c.) Neutralization (N) testing was done in Belém by the constant-serum, varying-virus technique, using infected mouse brain as virus source and antiserum of guinea pigs immunized with one subcutaneous injection of live virus. Serum-virus mixtures were incubated for one hour at 37°C prior to inoculation i.c. into baby mice. ISOLATION AND CHARACTERIZATION OF VIRUSES Aura and Una viruses were both first obtained from mosquitoes collected on human bait in the Instituto Agronômico do Norte (IAN) forest. The Aura prototype strain (AR 10315) was isolated from a pool of 99 Culex (Melanoconion) sp. collected during a nine Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A arthropod-borne viruses 165 days period in January-February, 1959, and the Una prototype (AR 13136) from a pool of 113 Psorophora ferox collected in September, 1959. By the end of August, 1961, 12 more isolations had been made from mosquitoes caught in the amazonian forests, two of strains apparently identical with AR 10315 and ten of strains apparently identical with AR 13136. A summary of these isolations is given in Table 1. Table 1 – Strains of Aura and Una viruses isolated from mosquitoes collected in the Amazon valley, 1959-1961 Mosquito source* Date of Virus strain Nº in Species pool Place collected isolation Aura AR 10315 Culex (Melanoconion) 99 IAN forest Feb.,1950 AR 24499 Aedes serratus 83 IAN forest Oct.,1960 AR 34049 Aedes serratus 61 IAN forest Aug.,1961 Una AR 20684 Aedes serratus 26 Tefé, Amazonas June,1960 AR 20690 Psorophora albipes 60 Tefé, Amazonas June,1960 AR 30826 Psorophora ferox 83 Km-94** May,1961 AR 30827 Psorophora ferox 83 Km-94 May,1961 AR 30856 Psorophora ferox 101 Km-94 May,1961 AR 30942 Psorophora ferox 77 Km-94 May,1961 AR 31557 Psorophora ferox 31 IAN forest May,1961 AR 32196 Psorophora ferox 125 Km-94 June,1961 AR 32978 Aedes leucocelaenus 67 Km-94 July,1961 * All mosquitoes were collected on human bait at ground level. ** Km 94 of the Belém-Brasilia highway, Pará. Aura virus had low pathogenicity for the mouse on primary isolation. The prototype strain was obtained from the only sick infant in a group of six inoculated with the original mosquito suspension. This animal was discovered with signs of central nervous system disturbance on the 7th day, and the brain was passed to another group of three day old mice. Two of the six infants in this passage survived, but virus was isolated from the other four which became sick and were sacrificed on the 5th, 7th and 9th days after inoculation. The material harvested on the 9th day, however, produced illness in only one of the six mice to which it was passed. By the 4th passage the average survival 166 Memórias do Instituto Evandro Chagas: Arbovírus time in infant mice inoculated either i.p. or i.c. had become established at about three days, while weanling mice showed no signs of illness. At this level the virus was shown to be filtrable through a Seitz EK pad. The two other isolates of Aura virus, obtained in October, 1960, and August, 1961, were likewise slow in becoming adapted to mice. By contrast, the Una strains were readily established in mice. The prototype strain produced sickness or death in all six infant mice on the 6th day after inoculation of original material, and was killing in three days at the 2nd passage. Filtration, pathogenicity for infant mice by the i.p. as well as i.c. route and resistance of weanling mice to i.c., i.p. and subcutaneous injection were demonstrated at the 3rd passage level. In 8th passage mouse brain, both prototype viruses titered between 8.3 and 8.5 log LD50. Neither prototype strain is pathogenic for guinea pigs by the subcutaneous route. Guinea pigs inoculated by the i.c. route survived and showed no visible signs of illness but did develop antibodies. One Aura and four Una strains were reisolated from original suspensions of mosquitoes in bovine albumin diluent stored at -75°F. Five other attempts to reisolate Una strains from original suspensions were unsuccessful. IDENTIFICATION OF VIRUSES In initial CF studies in Belém, antigens prepared from the Aura and Una prototype strains failed to react with sera against any of the arthropod-borne viruses previously isolated by this laboratory, including Venezuelan Equine Encephalitis (VEE), Eastern Equine Encephalitis (EEE) and Mayaro – the only three members of Casals’ group A then known to be indigenous to Brazil. In HI tests, however, both antigens were inhibited by a group A hyperimmune serum prepared from the three viruses mentioned, but not by immune sera against groups B, C and Guamá or ungrouped viruses of Brazil. Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A arthropod-borne viruses 167 Reciprocal cross-testing of the Aura and Una prototype strains and VEE, EEE and Mayaro by CF, HI and N techniques showed that the new isolates were distinct from each other and also differed significantly from the three known viruses. The Una strain exhibited some cross-reaction with Mayaro antiserum by all three tests. A summary of the N test results is given in Table 2. In HI tests carried out in the Rockefeller Foundation Virus Laboratories, New York, the two new agents were tested with immune sera against ten group A viruses. As shown in Table 3, the isolates were easily distinguishable both from each other and from the known viruses. Aura antigen was, however, inhibited to some degree by Western Equine Encephalitis and Sindbis antisera, while Una antigen again cross-reacted with Mayaro as well as with AMM 2021 antisera. The crossings of the two antigens with a VEE antiserum are considered negligible in view of the serum’s extremely high homologous titer. Table 2 – Neutralization tests with Aura and Una prototype strains and three group A viruses, log neutralizing indices Virus Serum Aura Una Mayaro EEE VEE Aura 1.9 0* 0 0 0 Una 0 2.7 0 0 0 Mayaro 0 1.9 > 3.5 00 = EEE 0 0 0 2.8 0 VEE 0 0 0 0 2.8 *0 = < 0.8 = Table 4 gives the results of HI tests in which one Aura and two Una immune sera were reacted against the homologous antigens and antigens from 11 group A viruses. The immune sera, which in all but one instance were derived from guinea pigs given a single i.c.