Fourth Army Area Medical Laboratory, Brooke Army Medical Center, Fort Sam Houston, Texan

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1 VIRUS OF BATS ANTIGENICALLY RELATED TO GROUP B ARTHROPOD-BORNE ENCEPHALITIS VIRUSES LT. COL. KENNETH F. BURNS, VC, USA, COL. CHARLES J. FARINACCI, MC, USA, AND DOROTHY F. SHELTON, B.A. Fourth Army Area Medical Laboratory, Brooke Army Medical Center, Fort Sam Houston, Texan A survey of bats was begun early in 195 at Brooke Army Medical Center, Fort Sam Houston, Texas, owing to the discovery of encephalitis among bats (Tadarida brasiliensis mexicana) that roost beneath the tile roofs of structures of Spanish architectural design. The encephalitis was manifested by deranged behavior, muscular tremors, urinary incontinence, and paretic changes. Thousands of bats died. During the survey of this colony of bats for naturally occurring rabies, on various occasions we isolated neurotropic viruses (other than rabies) from the salivary glands of Mexican free-tailed bats. 3 Initial studies of host susceptibility and pathogenicity for mice suggested the possible relation of the isolates. Previously we reported these viral isolates as antigenically related to the virus of St. Louis encephalitis. The present paper deals with continued attempts to establish the identity of these agents, with particular emphasis on isolate 1] -19. Our studies provide evidence that the salivary gland virus of bats, in addition to being antigenically related to St. Louis encephalitis virus, is also related to other members of the Group B arthropod-borne viruses, rather than having a specific relation to any one virus in the group. Further, it is probably a distinct, new entity. ISOLATION OF VIRAL AGENTS Brain. From the brains of 335 necropsied bats, 9 viral isolates were obtained in white Swiss mice. 1 ' 2 These isolates were identified as the virus of rabies by the standard intracerebral neutralization technic. Tests with antigen from infected mouse brain and known rabies-immune horse serum demonstrated that the immune serum neutralized its homologous virus as well as the newly isolated bat strains. Salivary gland. In addition to the confirmed isolations of rabies virus, other viral agents (strains , , , and 11-19) were recovered from the salivary glands of 38 encephalitic bats (T. brasiliensis mexicana). No strains of bat salivary gland virus were isolated from those animals in which rabies virus was recovered; furthermore, we did not recover the unknown virus from the brains of encephalitic bats in which the salivary glands were positive. The virus isolated from salivary glands of bats has an incubation period of 5 to Received, March 13, 1956; revision received, February 2, 1957; accepted for publication, February 6. Col. Farinacci is Commanding Officer, Lt. Col. Burns is Chief of the Virology and Veterinary Branches, and Mrs. Shelton is Supervisory Bacteriologist, Virology Branch. 257 Downloaded from on 3 November 217

2 258 BURNS ET Ah. Vol days when inoculated intracerebrally in mice. Suspensions of mouse brain with virus (LD ) were inoculated intracerebrally (.3 ml.) into bats (7'. brasiliensis mexicana) obtained from Bracken Cave, Texas. These bats had been confined in isolation at this laboratory for 3 days prior to inoculation. The inoculated bats developed a clinically apparent encephalitis 6 days postinoculation. Suspensions of salivary gland from these moribund bats killed mice in 5 to 6 days. Brain material from the same bats produced manifestations of the disease when inoculated intracerebrally into mice. With the exception of mice and bats, studies of host susceptibility in dogs, rabbits, hamsters, guinea pigs, arid goats were negative when the animals were challenged by intracerebral, intraperitoneal, and intramuscular routes. The single intracerebral or intramuscular inoculation of dogs, rabbits, hamsters, guinea pigs, and goats, in order to determine the host range, failed to elicit an antigenic response, as measured for complement-fixing or neutralizing antibody. Hyperimmune serums to each of the isolates were prepared in rabbits and guinea pigs, following intraperitoneal inoculations of virus (1 6 mouse LD 5 ). Complement fixing titers ranged from 1:8 to 1:256, and a log neutralization index of. for the homologous virus was demonstrated. The bat virus (isolate 11-19) required no adaptive procedures to establish its growth character in chick embryos. It proliferated in 1- and 11-day-old chick embryos after inoculation" of the chorioallantoic membrane, and was carried through successive passages, attaining a maximum titer of 1~ 6 LD5 when titrated intracerebrally in mice. The only alteration in physical appearance observed in the chick embryo was the thickening of the chorioallantoic membrane after to 6 days of growth. Growth of the virus did not cause death of the embryos. A 1 per cent suspension of harvested membranes was infective for 3-week-old mice when inoculated intracerebrally in amounts of.3 ml. per animal. The incubation period was to 6 days, with paralysis and prostration resulting. IDENTIFICATION OF BAT SALIVARY GLAND VIRUS Complement-fixation tests were performed essentially as the procedure outlined by Casals and Palacios, 6 with overnight incubation of the serum-antigencomplement mixtures. Reciprocal complement-fixation tests suggest that the bat virus shares some antigen in common with St. Louis encephalitis virus (Table 1). Thus, specific guinea pig antiserums for St. Louis encephalitis virus (SLE), inactivated at 56 C. for 3 minutes, consistently fix complement (low order, 1:) with the bat viral antigens. This reaction is not reciprocal, inasmuch as guinea pig hyperimmune serums prepared against the salivary isolates from the bats do not fix complement in the presence of SLE antigen. Hyperimmune guinea pig serums for eastern, western, and Venezuelan equine encephalitis, lymphocytic choriomeningitis, rabies, Japanese B encephalitis, and herpes simplex virus fail to fix complement in the presence of bat viral complement-fixing antigens. Neutralization tests were performed with undiluted serum and decimal dilu- Downloaded from on 3 November 217

3 March 1957 VIRUS OF BATS 259 TABLE 1 TITERS OF COMPLEMENT-FIXING ANTIBODIES IN GUINEA PIG SERUMS HYPER IMMUNE TO ST. LOUIS ENCEPHALITIS (SLE) AND VIRAL ISOLATES FROM BAT SALIVARY GLAND Complement-Fixation Antigens Guinea Pig Serums SLEt * * H1-19* Normal mouse brain antigen Anticomplementary control SLE Hyperimmune Hyperimmune Hyperimmune Hyperimmune Normal 16 S S S S 16 The figures indicate titers as the reciprocal of the dilution, and a zero indicates a titer less than 1:. * Prepared from the fifth passage of a suspension of mouse brain, according to the procedure outlined by Casals (J. Immunol., 56:337-31,197). Four units were used in the tests. f Commercial antigen. tions of virus, according to (1) the method recommended by the Neurotropic Virus Disease Commission, as quoted by Paul, 9 or (2) by the method outlined by Johnson, 8 in which constant amounts of virus (2 LD 6 per.3 ml.) were used with undiluted and 1-fold dilutions of serum. The bat virus is neutralized by St. Louis encephalitis hyperimmune rabbit serum. Cross-neutralization tests indicate that this reaction is not reciprocal, inasmuch as high-titered immune rabbit serums prepared against the isolate from bats (11-19) do not neutralize the virus of St. Louis encephalitis. In addition, the bat virus is not neutralized by the antiserums of western equine encephalomyelitis, eastern equine encephalomyelitis, lymphocytic choriomeningitis, or rabies. Similar results were obtained by personnel of Walter Reed Army Institute of Research, Division of Communicable Diseases, who also found no serologic relation to encephalomyocarditis virus (Table 2). Studies reported to us by Dr. W. McD. Hammon, University of Pittsburgh, indicate that, in addition to the bat virus being neutralized by immune St. Louis encephalitis rabbit serum, a distinct crossing with Murray Valley encephalitis, Japanese B encephalitis, and West Nile encephalitis antiserums is observed (Table 3). Dr. Hammon also reported that rabbit antiserum which neutralized. logs of homologous bat virus (11-19) also neutralized 2. logs of Ntaya virus. In his experience such a potent antiserum did not neutralize the viruses of Japanese B encephalitis (JBE), Murray Valley encephalitis (MVE), St. Louis encephalitis (SLE), West Nile (WN), and epidemic keratoconjunctivitis (Sanders) (EK). The unilateral reactions (i.e., nonreciprocal) obtained by cross-neutralization tests with NW, MVE, SLE, and JBE viruses, and the protective activity displayed for Ntaya virus by the bat virus hyperimmune serum, offer further evidence of the interrelation of bat virus to the B Group of viruses. By means of using procedures outlined by Chanock and Sabin 6 for the prepara- Downloaded from on 3 November 217

4 26 BURNS ET AL. Vol. 27 TABLE 2 SEROLOGIC IDENTIFICATION OF BAT VIRUS (11-19) BY CROSS-NEUTRALIZATION TESTS* Serums Log Neutralization Index vs. Homologous Virus Titer Virus in Respective Serum Log Neutralization Index of Serums vs SLE Rabbit Normal Immune St. Louis immune Western equine immune Eastern equine immune Lymphocytic choriomeningitis immune Encephalomyoearditis immune Horse Rabies immune (titer of 1:S vs. 1 LD M ) > >. 1Q-V.5 <1" >io- 6 - >io- i -' > 1Q-6. MO- 6 -* >1 _ «- >. l.s. 2. t : t' t t t * These tests were performed by the staff of the Walter Heed Army Institute of Research, Division of Communicable Diseases, Walter Reed Army Medical Center, Washington, D. C. f Not tested. TABLE 3 IDENTIFICATION OF BAT VIRUS (11-19)* BY INTRACEREBRAL NEUTRALIZATION TESTJ MOUSE Hyperimmune Serum Log LDso Log Neutralization Index Homologous Log Neutralization Index Western equine encephalitis Eastern equine encephalitis Japanese B encephalitis (Nakayama) West Nile encephalitis Epidemic keratoconjunctivitis (C1191) (Sanders) St. Louis encephalitis (Webster) Murray Valley encephalitis Normal rabbit serum < > * Fifth passage mouse brain material. f The authors are indebted to Dr. W. McD. Hammon, Head of the Department of Epidemiology and Microbiology, University of Pittsburgh, Graduate School of Public Health, Pittsburgh, Pennsylvania, for the results of tests reported in this table. tion of the hemagglutinin of St. Louis encephalitis virus, we succeeded in obtaining a potent hemagglutinin from the brains of suckling mice that were infected by fifth-passage material containing bat virus. This alkaline-buffered saline extract (at the time of preparation) reacted with chick erythrocytes at C, 25 C, and 37 C, within a range of ph from 6. to 6.6 (Table ). The optimum ph for reaction of this freshly prepared hemagglutinin seemed to be 6., with titers of 1:256 to 1:512. It was noticed that, if titrations of these antigens Downloaded from on 3 November 217

5 March 1957 VIRUS OF BATS 261 fs in Storage H i TABLE INFLUENCE OF TEMPERATURE OF INCUBATION AND I-H ON HEMAGGLUTINATION Hemagglutinin Incubated with Erythrocytes at C. 25 C. 37 C. C. 25 C. 37 C. C. 25 C. 37 C. OF CHICK ERYTHROCYTES BY BAT ANTIGEN (11-19) STORED AT C Reciprocal Hemagglutination Titer at Indicated ph 6. > S >12 > > > S S ph Range for Reaction * ph for Maximum Titers * The broadening of the ph range for reaction to the acid side on ripening, as well as the marked acid shift in the ph for maximum titers, is in contrast to that reported for the St. Louis encephalitis hemagglutinin, which exhibits a shift to the alkaline side during a similar period of storage. (which were positive at C.) were shaken and then incubated at 37 C, a positive reaction occurred with a titer that was equal, or almost equal, to that observed with direct incubation at C. If tests with antigens that yielded a positive reaction at 37 C. were shaken and incubated at C, the titers of the tests remained practically unchanged. Additional tests conducted after storing the antigen at C. for 15 and 35 days, in order to stud}' the influence of temperature of incubation and ph, demonstrated that maximal hemagglutination occurs at ph 6. to 6.3 when the antigen is diluted and incubated with erythrocytes at C. or 25 C. When incubated at 37 C, the optimal ph range seems to be 6.1 to 6.3. Although SLE hemagglutinin is active at this ph (6. to 6.), it is difficult to demonstrate at 37 C. Further dissimilarity is noted in the fact that the hemagglutinin of bat virus has one of the narrowest ph ranges in the particular zone of effectiveness of any neurotropic virus described. Adjustment of the ph of the diluent for the antigen, by as little as.3 of a ph unit in either the acid or alkaline direction, results in great depression, or complete masking of activity (Table ). Hemagglutination-inhibition tests (Table 5), performed in essentially the same manner as that described by Chanock and Sabin 7 for St. Louis encephalitis virus, indicate that heniagglutinating antigens prepared from each of 3 bat salivary gland isolates are inhibited by St. Louis encephalitis hyperimmune guinea pig serums (1:2 to 1:), and, in addition, by convalescent human serum (1:8 to.1:32). The St. Louis encephalitis human convalescent serum was obtained from a patient in whom the diagnosis was established by complement fixation tests (1:32) performed by the staff of the Texas State Bureau of Laboratories. The patient was 1 of many who were ill in an epidemic (identified as St. Louis encephalitis) that occurred in the Texas 1-Jio Grande Valley in 195. Downloaded from on 3 November 217

6 262 BURNS ET Ah. Vol. 27 TABLE 5 INHIBITION OP BAT VIRUS HEMAGGLUTINATING ANTIGENS BY HYPERIMMUNE GUINEA PIG SERUMS Hemagglutinin* Acetone Precipitated Fraction SLE Normal so SO so 16 2 (32) 2 (SO) (16) () () () The figures indicate the titer as the reciprocal of the highest dilution of serum that resulted in complete inhibition of hemagglutination. A zero indicates no inhibition by a 1:1 dilution of serum. The figures within parentheses indicate results obtained with serums from normal and convalescent human patients. * Sixteen units of the bat hemagglutinin were used in the tests. TABLE 6 INHIBITION OK HEMAGGLUTININ BY VARIOUS HYPERIMMUNE SERUMS Hemagglutination-inhibition Titers vs immune rabbit pool Japanese B encephalitis immune rabbit St. Louis encephalitis immune rabbit pool West Nile immune guinea pig pool hemagglutinin (2 units) 1:16 1: 1: 1: Homologous hemagglutinin ( to 8 units) 1:6 1:32 1:6 The titers indicate the highest dilution of serum that resulted in complete inhibition of hemagglutination. In addition, hyperimmune guinea pig serums for the 3 isolates from bat salivary glands j'ielded hemagglutination-inhibition titers of 1:8 to 1:16 when they were tested with their homologous hemagglutinating antigens (Table 5). These findings, coupled with comparable complement-fixation results obtained with and St. Louis encephalitis antigens (Table 1), suggest that the 3 isolates are,probably the same virus and that they are antigenically related to St. Louis encephalitis virus. The specificity of hemagglutinin was studied in hemagglutinationinhibition tests, employing hyperimmune.animal serums prepared against the bat salivary gland virus and those of West Nile, Japanese B encephalitis, and St. Louis encephalitis. These tests (Table 6) indicate (1) that serums prepared against the abovementioned viruses are inhibitory, and (2) that the homologous serum titer for was considerably higher than that of any of the heterologous serums {i.e., 1:16 compared with 1:). PATHOLOGIC ANATOMY The brains of experimentally inoculated mice are grossly normal. In some specimens the leptomeningeal blood vessels seem to be congested, with the hyperemia being more conspicuous in focal zones. Downloaded from on 3 November 217

7 March VIRUS OF BATS 263 Microscopically, the cerebellum, midbrain, and cerebrum of passage material from mice are characterized by an intact pia arachnoid, with focal infiltrations of lymphocytes about the vessels. Vascular congestion of the meningeal and intracerebral vessels is observed, and focal collections of microglia and of polymorphonuclear leukocytes are also noted. The cellular architecture of the neurons manifests variable degrees of chromatolysis in focal regions, and minimal evidence of satellitosis is noted. Stovall-Black 1 stains fail to reveal the presence of intranuclear or intracytoplasmic inclusion bodies. DISCUSSION AND SUMMARY While surveying bat populations in Texas, we isolated a neurotropic virus in mice, and succeeded in preparing a potent hemagglutinin (> 1:12) for this isolate from infected brains of suckling mice. The capabilities of this virus to yield a hemagglutinin on the basis of (1) temperature of incubation ( C., 25 C, or 37 C.) and (2) rigid requirements of ph (6. to 6.6 for freshly prepared hemagglutinin, and 6. to 6.5 after 35 days of ripening) suggest that the antigen is derived from a virus that is a member of Casals' B group of arthropod-borne viruses. Hemagglutination-inhibition tests demonstrate that the hemagglutinin of bat virus is inhibited by antiserums for St. Louis encephalitis, Japanese B encephalitis, and West A T ile at low titers, but that it reacts best with its homologous antiserum. Results of reciprocal complement-fixation tests suggest that the bat virus shares an antigen in common with St. Louis encephalitis virus, as indicated by fixation of complement with St. Louis encephalitis antiserum in the presence of bat antigens. The fixation is of low order, and it is not reciprocal. No immunologic relationship between the bat virus and other neurotropic viruses (eastern equine encephalomyelitis, western equine encephalomyelitis, Venezuelan equine encephalitis, Japanese B encephalitis, lymphocytic choriomeningitis, or rabies) could be demonstrated by complement-fixation tests. Further evidence that the bat virus is immunologically related to, but distinct from, St. Louis encephalitis virus is obtained from the fact that this agent is neutralized by St. Louis encephalitis hyperimmune serum, without any evidence of reciprocal activity. Additional neutralization studies with the bat virus (11-19) demonstrate a distinct crossing with hyperimmune serums prepared against the viruses of West Nile, Murray Valley, and Japanese B encephalitis. Immune serums prepared against the bat virus neutralize the homologous virus, and, in addition, are protective against Ntaya virus; therefore, on the basis of neutralization tests, it would seem that there is a closer interrelation between Ntaya and the bat salivary gland viruses than that which exists for the viruses of WN, MVE, JBE, and SLE. Hyperimmune serums for western equine encephalomyelitis, eastern equine encephalomyelitis, Venezuelan equine encephalitis, lymphocytic choriomeningitis, encephalomyocarditis, and rabies viruses have no neutralizing effect against the bat virus. Inasmuch as the hemagglutination-inhibition test seems to be the more sensitive serologic technic for demonstrating antigenic relationship between broadly related arthropod-borne viruses, the failure to obtain identical titers of inhibition Downloaded from on 3 November 217

8 26 BURNS ET AL. Vol. 27 (with the antiserums prepared against the bat virus and those of St. Louis, West Nile, and Japanese B encephalitis) suggests that the bat salivary gland virus is probably a distinct, new entity. SUMMAKIO IN 1NTEHLINGUA In le curso de investigationes de populationes de vespertiliones in Texas nos isolava repetitemente ab le glandulas salivari del animales un virus que se provava neurotropic in muses. Nos succedeva a preparar uu potente hemagglutinina (< 1:12) pro iste isolato ex inficite cerebros de muses sugente. Le capabilitate del virus de producer un hemagglutinina super le base de (1) temperatura de incubation (, 25, o 37 C) e (2) rigide requirimentos de ph (6, a 6,6 pro hemagglutinina frescamente preparate e 6, a 6,5 post 35 dies de maturation) suggere que le antigeno es derivate ab un virus pertineiite al gruppo B de viruses portate per arthropodos (Casals). Es presentate datos que indica que il se tracta probabilemente de un nove e distincte entitate intra ille gruppo. REFERENCES 1. BURNS, K. F., AND FARINACCI, C. J.: Rabies in nonsanguivorous bats of Texas. J. Infect. Dis., 97: , BUKNS, K. F., FARINACCI, C. J., AND MURNANE, T. G.: Rabies in insectivorous bats of Texas. J. Am. Vet. M. A., 128: 27-31, BURNS, K. F., FARINACCI, C. J., MURNANE, T. G., AND SHELTON.D. F.: Insectivorous bats naturally infected with rabies in southwestern United States. Am. J. Pub. Health, 6: 1S9-197, BURNS, K. F., AND FARINACCI, C. J.: Virus of bats antigenicallv related to St. Louis encephalitis. Science, 123: , CASALS, J., AND PALACIOS, R.: The complement fixation test in the diagnosis of virus infections of the central nervous system. J. Exper. Med., 7: 9-26, CHANOCK,R. M., AND SABIN, A. B.: The hemagglutinin of St. Louis encephalitis virus: 1. Recovery of stable hemagglutinin from the brains of infected mice. J. Immunol., 7: 271-2S5", CHANOCK, R. M., AND SABIN, A. B.: Hemagglutinin of St. Louis encephalitis virus: properties of normal inhibitors and specific antibody; use of hemagglutination-inhihition for diagnosis of infection. J. Immunol., 7: , S. JOHNSON, HARALD N.: Rabies. In Viral and Rickettsial Infections of Man. Ed. 2, edited by T. M. Rivers, Philadelphia: J. B. Lippincott Co., 1952, p S.MADEL, J. E.: Serologic reactions in viral and rickettsial infections. In Viral and Rickettsial Infections of Man. Ed. 2, edited by T. M. Rivers. Philadelphia: J. B. Lippincott Co., 1952, p STOVALL, W. D., AND BLACK, C. E.: The influence of ph on the eosin methylene blue method for demonstrating Negri bodies. Am. J. Clin. Path., Tech. Supp., :.1-8, 19. Downloaded from on 3 November 217

SUSCEPTIBILITY OF SUCKLING MICE TO VARIOLA VIRUS

SUSCEPTIBILITY OF SUCKLING MICE TO VARIOLA VIRUS RONALD G. MARSHALL AND PETER J. GERONE U. S. Army Chemical Corps, Fort Detrick, Frederick, Maryland Received for publication December, 6 ABSTRACT MARSHALL,