Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A -borne 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 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 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 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 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 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 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 . 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. injection, showed a high degree of specificity; the only overlaps occurred between one of the Una sera and Chikungunya, Semliki forest, Mayaro and AMM 2021 antigens. 168 Memórias do Instituto Evandro Chagas: Arbovírus

Table 3 – Hemagglutination-inhibition tests with Aura and Una anti- gens and group A immune sera

Serum Antigen, 8 units Nº Virus Source Aura Una Homologous injections Aura Mouse 2 2560* 0** Guinea pig 1 160 0 Una Mouse 1 0 160 Guinea pig 1 0 640 Chikungunya Mouse 5 20 0 2560 Guinea pig 1 0 0 320 Semliki Mouse 5 0 – 1280+ Human – 0 0 5120 conv. Mayaro Mouse 5 20 160 2560 Guinea pig 1 0 20 2560 AMM 2021 Mouse 5 0 320 1280+ AMM 2354 Mouse 1 0 – 40 WEE Mouse 6 160 0 5120 Guinea pig 1 20 0 1280 Sindbis Mouse 3 80 0 1280 Mouse 1 20 – 320 EEE Mouse 5 10 – 1280 Human – 0 0 160 conv. VEE Mouse 6 40 40 20480+ Middelburg Mouse 5 0 – 2560 * 2560 = reciprocal of serum titer. ** 0 = no inhibition at 1:10, the lowest dilution used.

Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A arthropod-borne viruses 169

Table 4 – Hemagglutination-inhibition tests with Aura and Una immune sera and group A antigens

Serum* Antigen, 8 units Una Aura 12 Aura 160 0 0 Una 0 640 160 Chikungunya 0 40 0 Semliki 0 20 0 Mayaro 0 20 0 AMM 2021 0 20 0 AMM 2354 0 0 0 O’nyong-nyong 0** 0*** – WEE 0 0 0 Sindbis 0 0 0 EEE 0 0 0 VEE 0 0 0 Middelburg 0 0 0 * All immune sera except those tested with O’nyong-nyong antigen were prepared in guinea pigs given one injection. ** Mouse antiserum for Aura, two injections; homologous titer 1:2560. *** Mouse antiserum for Una, one infection; homologous titer 1:160.

Table 5 gives the results of CF tests between antisera for ten group A viruses and Aura and Una antigens, as well as homologous antigen and serum titers which were obtained simultaneously. A number of sera failed to react with either antigen, and the remainder did so only at one-eighth or less of homologous titer. The converse CF reactions are shown in Table 6. The immune serum against Aura virus, with an homologous titer of 1:256, reacted either not at all or to only an insignificant degree with the group A antigens. The immune serum against Una virus, also with an homologous titer of 1:256, reacted to only one-sixteenth of this titer with the antigens. These CF reactions are relatively specific, as would be expected of group A viruses. 170 Memórias do Instituto Evandro Chagas: Arbovírus

Table 5 – Complement-fixation tests with Aura and Una antigens and group A immune mouse sera

Serum Antigen Virus Nº injections Aura Una Homologous Aura 2 128/64* Una 3 8/8 256/64 Chikungunya 5 4/4 4/4 256/32 Semliki 6 0/0 8/16 256/64 Mayaro 5 0/0 4/4 64/8 AMM 2021 5 4/4 32/32 256/256 AMM 2354 5 0/0 0/0 64/16 WEE 6 4/8 0/0 128/64 Sindbis 4 0/0 0/0 128/32 EEE 5 0/0 0/0 256/128 VEE 6 4/4 0/0 512/128 Middelburg 5 4/4 0/0 64/16 * Reciprocal of serum titer/reciprocal of antigen titer.

Table 6 – Complement-fixation tests with Aura and Una immune sera and group A antigens

Antigen Serum* Aura Una Aura 256/128 8/8 Una 0/0 256/64 Chikungunya 0/0 16/16 Semliki 0/0 16/64 Mayaro 0/0 16/8 AMM 2021 4/4 16/256 AMM 2354 0/0 16/16 WEE 8/8 16/16 Sindbis 0/0 16/32 EEE 4/4 16/16 VEE 0/0 16/32 Middelburg 0/0 16/32 * Prepared in mice; two injections for Aura, three for Una. Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A arthropod-borne viruses 171

The results of the immunological tests thus indicate that Aura (AR 10315) and Una (AR 13136) are new viruses and should be classified as members of group A. The close relationship of the 12 other isolates to one or the other of the prototype viruses was established in CF testing. EPIDEMIOLOGY Prior to the isolation of the prototype strain of Aura virus in January-February, 1959, a total of 133,542 mosquitoes had been processed in the Belém Laboratory for possible virus isolation. Since that time a further 277,000 mosquitoes have been processed, of which approximately 62% were caught on human bait generally between 9a.m. and 3p.m., and 38% were caught on sentinel mice or other animal bait usually in the early morning hours. As stated earlier, the three strains of Aura and 11 strains of Una were all isolated from collections made with human bait at ground level. It may be noted here that three isolates now identified as Una virus have been obtained in Trinidad in the Bush Bush forest of the Nariva swamp. One strain was isolated in 1959 from Psorophora ferox and the other two in 1960 from sentinel mice and from Culex (Melanoconion) caudelli4. In a serological survey of residents of the Amazon valley, only one of 1,313 sera had HI antibodies to Aura virus, and these were in low titer. A total of 1,199 sera was tested against Una, of which 36 showed higher titers against this virus than against any of the other group A agents known in the region. These 36 were also positive for Una in N test. Nevertheless, it is not possible to state categorically that these reactions represent infections with Una because many of these sera also react with Mayaro by HI and N test, although to a lesser degree. If Una were not known, the reactions would be considered indicative of previous infection with Mayaro and, indeed, they may represent infection with yet a different, but related, virus. Of the remaining sera tested with 172 Memórias do Instituto Evandro Chagas: Arbovírus

Una, some had HI antibodies in higher titer to Mayaro than to Una, and in general these sera also showed a higher neutralizing index against Mayaro. In HI tests with sera from other in the Amazon region, one of 288 marsupial, one of 955 rodent and one of 48 horse sera inhibited Aura antigen in low titers, while sera from monkeys, edentates, bats, lizards, rabbits, carnivores, birds, frogs, cows and sheep were negative. HI antibodies against Una were found in the sera of one of 361 rodents, two of 48 horses and one of 98 cows, but not in sera from marsupials, edentates, bats, carnivores, birds and sheep. SUMMARY Two hitherto unknown viruses, for which the names Aura and Una are proposed, have been isolated 14 times from mosquitoes collected in forested areas near Belém, Brazil, during 1959 to 1961. The three strains of Aura were obtained from Culex and Aedes spp., and the 11 strains of Una from Psorophora and Aedes spp. Aura has been shown to be immunologically related to Sindbis and Western Equine Encephalitis viruses and Una to Mayaro and AMM 2021 viruses, all of group A. Three strains of Una virus have also been isolated in Trinidad. A small percentage of sera from residents of the Amazon valley show HI and neutralizing antibodies against Una. The forest hosts of these two viruses are unknown. REFERENCES 1. CAUSEY, O. R., CAUSEY, C. E., MAROJA, O. M., AND MACEDO, D. G., 1961. The isolation of arthropod-borne viruses, including members of two hitherto undescribed serological groups, in the Amazon region of Brazil. Am. J. Trop. Med. & Hyg., 10: 227-249. 2. FULTON, F., AND DUMBELL, K. R., 1946. The serological comparison of strains of influenza virus. J. Gen. Microbiol., 3: 97-111. Causey, O. R.; Casals, J.; Shope, R. E.; Udomsakdi, S. Aura and Una, two new group A arthropod-borne viruses 173

3. CLARKE, D. H., AND CASALS, J., 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod- borne viruses. Am. J. Trop. Med. & Hyg., 7: 561-573. 4. AITKEN, T. H. G., Trinidad Regional Virus Laboratory, 1960. Personal communication.

ACKNOWLEDGEMENTS Mosquito collections at Tefé, Amazonas, and km 94 of the Belém-Brasilia highway, respectively, were made by Lerio Gomes, National Museum of Brazil, and dr. Hugo Laemmert, Oswaldo Cruz Institute. The wild animal sera were collected in part by dr. Laemmert, and the domestic animal sera in part by dr. Paul Sutmoller, FAO, and dr. Antonio Carlos Vahia de Abreu, SPVEA. The authors are indebted to dr. Max Theiler for valuable assistance in the preparation of this manuscript. 174 Memórias do Instituto Evandro Chagas: Arbovírus