Epidemiologic and virological observations in the Tampa Bay area California Group Arboviruses in Florida and Report of a New Strain, Keystone Virus J. O. BOND, M.D., W. McD. HAMMON, M.D., A. L. LEWIS, D.V.M., G. E. SATHER, M.P.H., and D. J. TAYLOR, M.S. PRESENCE of members of the Cali¬ Center of the Florida State Board of Health. THEfornia group of arboviruses was first dem¬ The isolation of members of this arbovirus onstrated in Florida in 1963, when human group from mosquitoes collected during the serologic responses to the original California 1962 epidemic of St. Louis encephalitis in the encephalitis virus (CEV) prototype antigen Tampa Bay area was reported subsequently by BFS-283 (1) were found by the University of the Communicable Disease Center, Public Pittsburgh and the Encephalitis Eesearch Health Service (2). Extensive studies by the authors and CDC Dr. Bond is director, Dr. Lewis is chief virologist, investigators have shown that California group and Mr. Taylor is chief entomologist, Encephalitis arboviruses are the most commonly recovered Research Center, Florida State Board of Health, mosquito-borne virus agent in Florida. Others Tampa. Dr. Hammon is professor of epidemiology, have shown that they also occur over a wide and Miss Sather is a research associate, department geographic area of the southeastern (3), cen¬ of epidemiology and microbiology, University of tral, midwestern (4, 5), far northwestern (6), Pittsburgh Graduate School of Public Health, Pitts¬ and Pacific coastal (7) sections of the United burgh, Pa. States. Earlier reports of viral agents in the Epidemiologic studies were directed in part by California group from Trinidad (8), Africa Dr. Emily H. Gates, biological studies by Dr. Wil¬ (9), Czechoslovakia (10), Yugoslavia (ii),and liam L. Jennings, entomological identification by Canada (12) give a worldwide significance to Karen Meadows, serologic work by Florence Y. the small but rapidly growing serologically re¬ Lewis, and statistical analyses by Ingrid Baugh- lated group of arboviruses. man. all on the staff of the Encephalitis Research Our report summarizes virological, epidem¬ Center. iologic, and serologic studies of the California Public Health Service research grant A1-05504- group of arboviruses in the Tampa Bay area of 02,03 partly supported the studies in Florida. De¬ Florida from 1963 to 1965. In addition, a partment of the Army contract DA-49-193-MD- newly identified member of the group, the Key¬ 2042 and Public Health Service research grant stone virus, is reported. The name Keystone Al-02686 partly supported the studies at the Uni¬ was derived from a geographic location in the versity of Pittsburgh, which were sponsored by the Tampa Bay area where mosquitoes were col¬ Commission on Viral Infections, Armed Forces lected on the night of August 11, 1964. A Epidemiological Board. report of the detailed serologic studies of this Vol. 81, No. 7, July 1966 607 and other known strains of the California rhynchus comprise the most abundant species of group will appear in a separate publication. mosquito in the Tampa Bay area; however, in¬ Data have been presented in part (13). tensive efforts have not been made to trap it. The Aedes atlanticus tormentor species are more Methods commonly attracted to lighted traps than to baited traps, but the latter were not used exten- A comprehensive description of the methods sively in the surveillance program until 1964. used for studying these viruses by the Florida After initial isolation and identification of State Board of Health and the University of CEV strains by the Encephalitis Eesearch Pittsburgh has been published (14)- Briefly, Center laboratory in Tampa in 1963 and 1964, the 2- to 4-day-old suckling mouse has been the subsequent typing has been carried out at the primary host system used for recovering these University of Pittsburgh. We have sum¬ viruses from mosquitoes. Serologic studies for marized the following extensive studies, many evidence of infection in man and mammals have still in process. been performed with sucrose-acetone-extracted The eight viruses isolated in 1963 were identi¬ (15) antigens prepared from the prototype fied as closely related to, or identical with, the BFS-283 strain (1). Standard techniques trivittatus virus. All appeared to be similar have been used for detecting hemagglutination- (14). Ten of the viruses isolated in 1964, rep¬ inhibition antibodies with the microtiter adap¬ resenting six isolations from Aedes infirmatus tation of the method described by Clarke and and four from A. atlanticus tormentor are cur¬ Casals (15). Serum-neutralization antibody rently being studied. Some tests are incom¬ studies have been performed in the 3- to 4-week- plete; others have progressed sufficiently to old weanling mouse by using the intracere¬ draw certain conclusions. Preliminary comple- bral route and the constant-serum, varying ment fixation tests with crude (pH 9.0 borate virus-dilution technique. saline) antigens confirmed that all the 1964 strains belonged to the CEV group but indi¬ Identification of California Strains cated that all the isolates were not identical. Mouse immune serums and sucrose-acetone- a 32-month During periodbeginning January extracted antigens were prepared for each of 1963, approximately 500,000 mosquitoes were the 10 isolates, and complement fixation cross collected and tested for viruses as a the part of comparisons are being made with representa¬ routine surveillance program of the Encephali¬ tives of the CEV group. Some resembled the tis Eesearch in the area. Center Tampa Bay earlier Florida prototype identified as trivitta¬ In these 41 were found mosquitoes, pools posi¬ tus virus, but several appeared somewhat more tive for members of the California group. The closely related to the group prototype BFS-283 pools were distributed among at least four virus. BFS-283 antiserum reacted by comple- different Aedes species of mosquitoes, represent¬ ing 27,035 mosquitoes tested in 790 pools (table 1). The positive pools were collected through¬ Table 1. California group virus isolations out the entire Tampa Bay area where routine in Florida mosquitoes, 1963.64 surveillance trapping is done, and they show the ubiquitous distribution of fresh floodwater Aedes mosquitoes. Positive pools were recov¬ ered every month of the year except September, November, and December. A definite relation between virus isolations and the abundance of Aedes mosquitoes is apparent. In turn, this is related to rainfall. Mosquito-trapping techniques and the loca¬ tion of traps markedly influence the types of Aedes mosquitoes collected. Aedes taenio- 608 Public Health Reports Table 2. Complement fixation cross com¬ 10"4-5 logs. In complement fixation identi¬ parison of two 1964 isolates with the Cali¬ fication studies at the Encephalitis Eesearch fornia group BFS-283 and trivittatus Center, a BFS-283 antiserum titered 1: 64 in the viruses presence of a crude TA-26 antigen (pH 9.0 bo¬ rate saline). Additional comparative studies at the University of Pittsburgh demonstrated that the virus was readily neutralized by immune se¬ rums to the CEV group (neutralization in- dex=4.0), TB3-1 (neutralization index=3.6), trivittatus (neutralization index=3.4), La- Crosse (neutralization index=3.0), and Snow- shoe hare (neutralization index=3.2). These confirmed the isolate as a member of the CEV 1 Aedes infirmatus isolate. and further demonstrated the advan¬ 2 Aedes atlanticus tormentor isolate, the Keystone group, virus. tages of the complement fixation test for typing. 3 Florida prototype of trivittatus virus. These studies indicate the of at 4 Reciprocal of serum dilution showing complement joint presence fixation of more than 2. least two antigenic types of the CEV group in the Tampa Bay area during 1964: trivittatus ment fixation to higher titer with the Florida and the newly identified Keystone strain. At agents than with the trivitattus agents. the present time, the Keystone appears to rep¬ Two of the 1964 isolates have been compared resent another antigenic type not only for by complement fixation (table 2). TA-28 North America but for the world. (B64-2503.02) appears to be identical with the TB3-1 virus, the earlier Florida prototype re- sembling trivittatus. TA-26 (B64-5587.05) is Evidence of Clinical Infection in Man distinctly different from TB3-1 and also differs Since the major St. Louis encephalitis epi¬ significantly from BFS-283, the original CEV demic in the Tampa Bay area in 1962, routine prototype from California. surveillance for viral infections of the central At least 4 of the 10 viruses studied resemble nervous system in man has been carried out in the unique TA-26. Three of the four were iso¬ the area. In a population of approximately 1 lated from the A. atlcmticus tormentor and one million, 832 persons have been referred to the from the A. infirmatus mosquitoes. Thus the center for viral diagnostic studies over a 32- mosquito species does not necessarily correlate month period beginning January 1963. Of with the type of virus isolated. By simultane- these, 618 have received serologic tests for ous cross-box titration, three of the TA-26-like hemagglutination-inhibition antibodies to the agents, which have been completely compared, California complex group, using the BFS-283 appear to be identical. The newer member of antigen (table 3). Paired serums from each the CEV group from Florida, the Keystone, has been compared with other known isolates Table 3. Hemagglutination-inhibition anti¬ belonging in the group from the United States bodies to California group arboviruses in and abroad and is significantly different from 618 central-nervous-system surveillance all viruses so far available to us. cases, Tampa Bay area, Fla., 1963.65 The Keystone virus was isolated from a pool of 55 engorged A. atlanticus tormentor mosqui¬ toes collected in a stable trap, using an exposed donkey as bait.