Epidemiologic and virological observations in the Tampa Bay area

California Group Arboviruses in Florida and Report of a New Strain, Keystone

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 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, -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 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 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 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 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 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 was isolated from a pool of 55 engorged A. atlanticus tormentor mosqui¬ toes collected in a stable trap, using an exposed donkey as bait. The agent produced death or sickness of an entire litter of eight suckling mice on the fifth day after inoculation. Subsequent second and third mouse passages reduced the incubation to 3 to 5 The median period days. 1 August 1. lethal dose of the agent for weanling mice was 2 Acetone-extracted serums.

Vol. 81, No. 7, July 1966 609 suspected person have been tested simultane- ported in detail (16). The pertinent serologic ously. The large number of persons with an un- findings for both patients are given in table 4. changing titer in 1963 requires an explanation. Both patients were children. S.H. manifested In the early part of the study, human se¬ a relatively severe encephalitis syndrome from rums were routinely extracted with acetone which she recovered within a period of 3 according to the standard technique (4)* An months without apparent sequelae. M.H. had unusually high number of such serums pos- a relatively mild encephalitis and apparent full sessed hemagglutination inhibitors to the Cali¬ recovery within 2 weeks. A fairly prompt rise fornia antigens used in the test. In attempting and fall in hemagglutination-inhibition re¬ to confirm this unexpectedly large number of sponse occurred in both children within the first positive serums, the laboratory at the Univer¬ 3 months of illness. Complement fixation anti¬ sity of Pittsburgh subjected the serums to both bodies appeared more slowly and had not been acetone and kaolin extraction. Both laborato¬ detected in M.H. by the 94th day. Diagnostic ries found that the inhibitor was frequently rises in serum neutralization antibodies were present after acetone extraction but not after detected in both patients. Extensive serologic kaolin extraction. Serum-neutralizing anti¬ tests for other arbovira! agents and neurotropic body studies of these serums, using the BFS-283 viruses known to be present in the Tampa Bay prototype virus, demonstrated that the inhibi¬ area have been negative for both children. tor usually remaining after acetone extraction was most with the likely nonspecific. Serums Human inhibitor after kaolin extraction were also Serologic Surveys found to contain neutralizing antibody to the Another part of the continuing program of CEV group. All subsequent studies of human the Encephalitis Eesearch Center in Tampa has and mammalian serums at the Encephalitis Re¬ been serologic surveys for inapparent human search Center were therefore carried out after infection with various members of the arbovirus kaolin extraction. group. During the past 3 years, more than Three of the 369 surveillance patients tested 1,000 such serums have been subjected to the in 1964 and three of the 193 tested so far in 1965 hemagglutination-inhibition test, using th* have had low unchanging hemagglutination- BFS-283 antigen (table 5). All serums tested inhibition titers (table 3). However, in both and reported were kaolin extracted. The sur¬ 1963 and 1964, a single patient demonstrated a vey populations were predominantly healthy fourfold rise in antibody by the hemagglutina¬ residents of households selected for serial blood tion-inhibition test. One case has been re¬ samples after a major survey in 1962. The

Table 4. Two serologically confirmed cases of encephalitis due to a California group virus, Tampa Bay area, Fla., 1963-64

Titer to California antigen BFS-283 Patient Onset Serums collectedl Hemagglu¬ Comple¬ Neutraliza¬ tination in- ment fix¬ tion index2 hibition ation

3 <1:20 <1:4 <0.5 16 1:80 <1:4 3.0 S.H., age 11, white, female. Aug. 20,1963 30 1:640 1:16 3.5 120 1:20 1:32 2.8 4 1:10 <1:4 <.2 65 1:80 <1:4 2.7 M.H., age 2, white, male_ June 28, 1964 94 1:40 <1:4 2.6 280 1:10

1 Day after onset. 2 Neutralization index logio, 3- or 4-week-old mice, intracerebral route.

610 Public Health Reports Table 5. Human serologic surveys for California group arbovirus hemagglutination-inhibi¬ tion antibodies,1 Tampa Bay area, Fla., 1963.65

1 Antigen BFS-283 prototype. 2 Any titer of 1:10 or greater, kaolin-extracted serums.

Table 6. serologic surveys for California group arbovirus hemagglutination-inhibi¬ tion antibodies,1 Tampa Bay area, Fla., 1964.65

1 Antigen BFS-283 prototype. 2 Any titer of 1:10 or greater, kaolin-extracted serums. results of studies of two special groups, mentally positive titers, 26 were at the level of 1:10, 9 at retarded children and migrant laborers, are 1:20, and 2 at 1:40. Studies for confirmatory also reported in table 5. serum neutralization antibodies of 19 of these Using any titer of 1:10 or greater as a cri¬ serums were carried out at the Encephalitis Ee¬ terion for positives, the results show that 1 to 6 search Center. Of 11 serums reacting at 1:10 percent of the general population in the Tampa by the hemagglutination-inhibition test, 9 were Bay area possess hemagglutination-inhibition shown to have equal to or greater than 1.5 logs antibody to the CEV group. In the special protection. Of six reacting at the 1:20 hemag¬ population surveys, none of the 56 mentally re¬ glutination-inhibition titer level, all had equal tarded children possessed hemagglutination- to or greater than 1.5 logs protection. Of the inhibition antibody; however, 2 had serum neu¬ two reacting at the 1:40 level, both were shown tralization antibody. Thirteen percent of the to possess serum neutralization antibodies equal migrant labor group studied in 1965 had he¬ to or greater than 2 logs protection. Similar magglutination-inhibition antibody, a not un¬ results were found for a group of six other expected result due to their severe and pro¬ serums tested at the University of Pittsburgh longed exposure to mosquitoes in Florida and laboratories. elsewhere. Approximately 400 persons in the normal Of the total group of survey serums with household survey group have submitted blood

Vol. 81, No. 7, July 1966 611 219-039.66-3 samples annually for 3 years. No evidence of Health Service) is most commonly recovered conversion from negative to positive hemag¬ from A. infirmatus. The second serotype, the glutination-inhibition titers in any of the per¬ Keystone, appears to be distinct from other sons has been found over the 3-year period. known members of the CEV group and was most frequently found during this study in the Animal Serologic Survey A. atlanticus tormentor. Although our human and animal serologic Serologic tests have been performed on se¬ studies have been carried out only with the rums collected from 706 wild or domestic ani¬ BFS-283 antigen, detected infection with the mals of 20 different species. Studies were CEV group in both man and is con¬ performed as part of routine surveillance, as siderably less than that found in the Midwest well as special studies of the vertebrate hosts of and California, where similar extensive studies arboviruses known to be in the Tampa Bay have been done (4, 7). Only 2 clinical cases area. The largest number of mammalian se¬ were found after examining 618 patients, and rums were collected from species of cotton rats, from 1 to 6 percent of the normal human popu¬ horses, opossums, rats, and the house mouse. lation appear to have experienced inapparent A criterion of any hemagglutination-inhibition infection with these viruses sometime in the titer of 1:10 or greater on kaolin-extracted past. The current level of transmission is less serums was applied to the BFS-283 antigen. than 1 percent annually, based on serial obser¬ The species showing positive results are listed vations of more than 400 persons. Serologic in table 6. One of 23 rabbits tested had a titer evidence of infection in mammals is extremely of 1:40. Titers of 1:20 were found in one each low by the hemagglutination-inhibition test and of 149 horses and 267 cotton rats. Low titers most of the hemagglutination-inhibition posi¬ of 1:10 were also detected in nine serums from tives tested were not confirmed by the serum species of the squirrel, raccoon, opossum, and neutralization test. We do not know the signif¬ horse. Five of 12 reacting serums were sub¬ icance of this. It is possible that the duration jected to the serum neutralization test, using the of hemagglutination-inhibition antibodies is too BFS-283 virus. Insufficient quantities did not short for serologic survey work and that serum permit the testing of all 12 serums. Only one neutralization antibodies may reveal different of the five serums, that of the horse, with a prevalence rates in both man and animals. hemagglutination-inhibition titer of 1:20, con¬ tained serum neutralization antibody at a level Summary equal to or greater than 2 logs protection. The remaining four serums, all serum neutralization The 1963-65 ecologic studies for arboviruses negative, had hemagglutination-inhibition anti¬ in the Tampa Bay area by the Encephalitis Re¬ body titers of 1:10. search Center, Florida State Board of Health, have shown that the California encephalitis Discussion group arboviruses are the most commonly recovered viral agents from mosquitoes. Of These studies clearly demonstrate the abun¬ 27,035 Aedes mosquitoes tested in 790 pools dur¬ dance of arboviruses belonging to the CEV ing the period January 1963 to December 1964, group in the Aedes genus of mosquitoes in Flor¬ 41 pools were found to be positive. The trivit- ida. If appropriate trapping techniques are tatus-like strain was recovered most frequently used when and where these mosquitoes are and predominantly from Aedes infirmatus mos¬ abundant, viruses of this group can be readily quitoes. A newly identified strain, named the obtained throughout an entire area at any season Keystone, was obtained from the Aedes atlanti¬ of the year. These strains have demonstrated cus tormentor. at least two distinct serotypes. One serotype, Human disease related to California viruses closely related if not identical to the trivittatus by serologic tests has been uncommon. Two isolate (unpublished data of C. M. Eklund, di¬ patients with viral symptoms of the central rector, Rocky Mountain Laboratory, Public nervous system were identified in 618 tested.

612 Public Health Reports Human infection without history of central- (6) Newhouse, V. P., Burgdorfer, W., McKiel, J. A., nervous-system disease was found in 1 to 6 per- and Gregson, J. D.: California encephalitis virus: Serologic survey of small wild mam- cent of -the general population. The authors mals in northern United States and southern emphasized the importance of extracting human Canada and isolation of additional strains. serums with kaolin rather than acetone to detect Amer J Hyg 78: 123-129 (1963). specific inhibitor to BFS-283 antigen. (7) Gresikova, M., Reeves, W. C., and Scrivani, R. P.: The lower vertebrate source of the California California encephalitis virus, an evaluation of encephalitis group arboviruses in Florida its continued epidemic status in Kern County, California. Amer J Hyg 80: 229-234 (1964). remains unknown. Extremely low rates of (8) Spence, L., Anderson, C. R., Aitken, T. H. G., and hemagglutination-inhibition a n t i b o d y have Downs, W. G.: Melao virus, a new agent iso- been found in mammals. Preliminary serum lated from Trinidadian mosquitoes. Amer J neutralization tests suggest that most of these Trop Med 11: 687-690 (1962). are nonspecific. The year-round recovery of (9) Kokernot, R. M., et al.: Isolation of viruses from mosquitoes collected at Lumbo, Mozambique. the virus from mosquitoes, however, suggests its I. Lumbo virus, a new virus isolated from continual presence in a common host. Aedes (Slusea) pembaensis Theobald. Amer JTropMed 11: 678-682 (1962). and V.: The REFERENCES (10) Bardos, V., Danielova, Tahyna virus; a virus isolated from mosquitoes in (1) Hammon, W. McD., Reeves, W. C., and Sather, Czechoslovakia. J Hyg Epidem (Praha) 3: G.: California encephalitis virus, a newly de- 264-276 (1959). scribed agent. II. Isolation and attempts to (11) Likar, M., and Casals, J.: Isolations from man identify and characterize the agent. J Im- in Slovenia of a virus belonging to the Cali- munol 69: 493-510 (1952). fornia complex of -borne viruses. (2) Chamberlain, R. W., Sudia, W. D., Coleman, P. Nature (London) 197: 1131 (1963). H., and Beadle, L. D.: Vector studies in the (12) McKiel, J. A., et al.: Viruses of California en- St. Louis encephalitis epidemic, Tampa Bay cephalitis complex in indicator rabbits. area, 1962. Amer J Trop Med 13: 456-461 Amer J Trop Med 15: 98-102 (1966). (1964). (13) Hammon, W. McD., and Sather, G. E.: History (3) U.S. Public Health Service: Summary of arbo- and recent reappearance of viruses in the Cali- virus encephalitis in the United States for the fornia encephalitis group. Amer J Trop Med year 1964. Morbidity and Mortality Weekly 15: 199-204 (1966). Report, vol. 14, No. 29, Communicable Disease (14) Lewis, A. L., et al.: Isolations of a California Center, Atlanta, Ga., July 24, 195, pp. 246-248. group arbovirus from Florida mosquitoes. (4) Thompson, W. H., and Evans, A. S.: California Amer J Trop Med 14: 451-455 (1965). encephalitis virus studies in Wisconsin. (15) Clarke, D. H., and Casals, J.: Techniques for Amer J Epidem 81: 230-244 (1964). hemagglutination and hemagglutination- (5) U.S. Public Health Service: Summary of Cali- inhibition with arthropod-borne viruses. Amer fornia encephalitis cases in Wisconsin, Ohio J Trop Med 7: 561-573 (1958). and Indiana. Morbidity and Mortality Weekly (16) Quick, D. T., et al.: California encephalitis Report, vol. 13, No. 47, Communicable Disease virus infection; a case report. Amer J Trop Center, Atlanta, Ga., Nov. 27, 1964, pp. 414-415. Med 14: 456-459 (1965).

Vol. 81, No. 7, July 1966 613 Vaccine Against Rubella THE FIRST EFFECTIVE experimental cultures over a 2-year period. Their study of vaccine against rubella has been developed by the biological characteristics of each virus pas¬ Dr. Harry M. Meyer, Jr., and Dr. Paul D. Park- sage level showed high-passage virus to be dis- man of the Division of Biologics Standards, tinctly different from low-passage "virulent" National Institutes of Health, Public Health virus. The high-passage material caused a Service. rapid cytopathic effect and other distinctive changes in certain types of tissue cultures un- Working with the Department of Pediatrics the virulent virus. It also induced of Arkansas Medical School affected by of the University the production of increased amounts of inter- and the Arkansas Children's Colony, near Con- feron when in tissue cultures. the vaccinated propagated way, pediatrician-virologists High-passage virus of the 77th passage level eight rubella-susceptible children in the pilot immunized rhesus without clinical trial. The children immu¬ (HPV-77) monkeys developed viremia or to uninoculated contacts. with no fever or rash and spread nity accompanying The HPV-77 strain was considered suffi- their with eight rubella-susceptible playmates, modified to be used in a clinical whom shared a remained unin- ciently trial, they cottage, and the vaccine was fected. The same results were obtained when experimental prepared, the same standards and tests that an additional 26 children were inoculated and using safety to the of live attenuated vac¬ were 22 uninoculated apply production in contact with playmates. cines for measles and poliomyelitis. The atten¬ In searching for a strain of the rubella virus uated rubella virus strain is being made avail¬ that would confer long-term immunity, Meyer able to university investigators and a number and Parkman subjected the virus to 77 passages of pharmaceutical companies that are working in primary African green monkey kidney cell toward a rubella vaccine.

Dr. Meyer (front) and Dr. Parkman (rear) with students of Arkansas Children's Colony and nurse Mrs. Bill Holloway

614 Public Health Reports