THE IDENTIFICATION OF NEGISHI VIRUS A PRESUMABLY NEW MEMBER OF RUSSIAN SPRING-SUMMER ENCEPHALITIS VIRUS FAMILY ISOLATED IN JAPAN*

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1 Jap. J. M. Sc. & Biol., 14, 51-59, 1961 THE IDENTIFICATION OF NEGISHI VIRUS A PRESUMABLY NEW MEMBER OF RUSSIAN SPRING-SUMMER ENCEPHALITIS VIRUS FAMILY ISOLATED IN JAPAN* TAKESHI OKUNO, AKIRA OYA AND TOSHIKO ITO Department of Virology & Rickettsiology, National Institute of Health, Tokyo (Received: March 20th, 1961) In the summer of 1948, a virus was isolated in Tokyo City from the cerebrospinal fluid of a patient who was clinically diagnosed as Japanese B encephalitis. The isolation was done by Ando et al.(1952) employing the intracerebral (i. c.) inoculation into adult mice. Since the virus showed no immunological overlap with Nakayama strain of Japanese B, it was thought that this might be a mutant of Japanese B. Later in the same year, another successful isolation was made again from an autopsied patient's brain which was termed as Strain K-13 (Ando et al., 1,952). According to marked similarities in biological and i mmunol ogical characteristics, Negishi and K-13 have been considered to be identical with each other. A case of laboratory infection occurred in 1950, which was manifested not with an overt encephalitis but with fever. Subsequently there have been a number of studies (Honda, 1951) directed toward the serological identification of this virus; however, most of them ended eventually in an equivocal or inconclusive results. Hence, in our labora tory, it was decided to carry out the identification once again with the latest serological techniques. The present paper decribes how this virus was identified as a prsumably new member of tick-borne arbor virus. This is the first report demonstrating the presence in Japan of virus of the Russian Spring-Summer Encephalities (RSSE) family. MATERIALS AND METHODS Viruses: 1) Negishi: Since the virus has already undergone a number of i.c. passages through adult mice, the level of passage was gunknown h plus five in suckling mice by the i.e. route. The LD50 usually seen were: and in adult mice when given by the i. c. and intraperitoneal (i) p.) route, respectively, and in suckling mice inoculated i.e. The average incubation period in suckling mice inoculated i.e. was 3 to 4 days with more than 1,000 LD, and 5 days with 100 LD. The virus behaved as a typical arbor virus, when tested against desoxycholate (Theiler, 1957) or protamine sulphate (Warren et al., 1949). 2) Others: Other viruses employed for the preparation of antigens or antisera are listed in Table 1 with a reference to the passage levels and the origins from which they were derived. Preparation of antigen: Since most of the viruses under study belong to Group B arbor virus (Casals, 1957), all the antigens were prepared according to Clarke and Casals (1958). Acetone ether extraction technique was employed in most of the cases with a slight modification of the original method i.e., the resuspension of dried powder was performed so as to yield * Aided in part by Grant RE supplied to this Institute from the Rockefeller Foundation, U.S.A.

2 52 OKUNO et al. Vol. 14 Table 1. Histories of the viruses employed for the serological identification of Negishi virus. * The Rockefeller Foundation Virus Laboratories, New York. ** Isolated by McLean and Donohue (1959). See also Casals (1960). *** Isolated by Matsuyama et al.(1960). a 25% antigen. In the case of Negishi, various techniques were tried in comparison as shown in the text. All the antigens were freeze-dried in vacuo and kept at -20 Ž for storage. Preparation of antiserum: Guinea pigs and, more frequently, mice were used for an im munization. The general principle varied according to the biological property of the virus concerned. In the case of RSSE, Powassan and Negishi viruses, the initial immunization of mice was made intraperitoneally with a vaccine prepared from a 20% suspension of infected suckling mouse brain made in buffered saline of ph 9.0 (BS 9.0), which had been inactivated b y mixing with an equal volume of 0.1% ƒà-propiolactone also made in BS 9.0 and placing at 37 Ž for an hour. The adoption of ƒà-propiolactone for inactivation of various animal viruses has been described elsewhere (LoGrippo, 1958, 1960). Immunization of guinea pigs was carried out with a fresh live virus vaccine throughout Except three viruses above cited either guinea pigs or mice were immunized intraperitoneally with live virus vaccine 3 to 5 times with one to two weeks' interval. The same schedule of vaccination was applied to the animals which had been initially immunized with ƒà-propiolactone vaccine a couple of week previously. The animals were bled partially a week following one to two immunizations or bled to death a week after 3 to 5 immunizations. The former type serum was tentatively termed as ginitial h serum, and the latter as ghyperimmune h serum. The degree of immunization was expressed by the number of injections which will appear later in the text. The sera were dispensed into a number of short pyrex tubes where necessary, and then stored at -20 Ž until use. Hemagglutination (HA) and hemagglutination inhibition (HI) techniques: These are the one which was originated by Clarke and Casals using lucite trays (1958). A slight modification was made for the HA-HI diluent by employing egg albumin instead of bovine plasma albumin in the same concentration. The egg albumin which is currently in use in our laboratory was prepared in the following manner. Four grams of egg albumin soluble (Difco) were suspended in 100 cc of BS 9.0 with the aid of a mortar and a pestle. The resultant was a stock 4% egg albumin (EA) which was subsequently diluted to make a 0.4% solution after a light centrifuga tion by mixing one part of the supernate and 9 parts of BS 9.0. Freshly prepared 0.4% EA is usable without an appreciable amount of flocculation for at least 3 days. The stock EA can be stored in a 4 Ž refrigerator over a couple of months. Goose cells were employed exclusively in a concentration which gave an optical density of in a Coleman Junior Photoelectric Spectrophotometer at wave length 490 mte and in tubes of 10 mm in diameter. All sera were treated with acetone (First class chemical reagent, Japan) and additionally absorbed with goose cell for HI tests..

3 1961 NEGISHI VIRUS 53 Complement fixation test (CFT): The CFT was carried out two-dimensionally by dilutions of a serum and an antigen. The methods were essentially the same as what has been exclusively employed in the Rockefeller Foundation Virus Laboratories, New York, as a modification of Fulton-Dumbells' drop technique (1949) on disposo CF trays*. Each of an inactivated serum and an antigen was delivered in one drop into disposo-trays prior to the addition of 1.7 to 2.2 units of guinea pig complement in two drops. The antigenantibody-complement mixture was allowed to stand in a 4 Ž refrigerator overnight and followed by the addition of two drops of hemolytic system which was composed of approximately twenty units of hemolysin and 2.5% sheep cell suspension. The dropping needles had been assayed prior to the test so as to deliver one cc of the diluent in 50 }2 drops. Veronal buffered saline was employed as a diluent according to the description by Fulton and Dumbell (1949). Complement fixing endpoints were the highest dilution of serum and/or antigen which showed 25% or less hemolysis. RESULTS Efficiencies of HA Antigen Production by Various Techniques A batch of infected suckling mouse brains of passage 6 was subjected to a compara tive test on the efficiency of antigen production by various techniques. Table 2 illust rates the results obtained in a single HA test, indicating that crude alkaline aqueous and acetone ether antigens yielded poor hemagglutinin (HAnin) titer whereas the best production was obtained with sucrose acetone antigen treated additionally with protamine sulphate. Of interest was a marked degree of improvement in the HA titer by the use of protamine sulphate irrespective of the technique for antigen extraction. It may be justified to postulate that this antigen source has at least two distinct inhibitors, one of which is eliminated by acetone ether and the other by protamine sulphate. Relatively poor yields of acetone ether preparation may be attributable to the drastic extraction with ether since the sucrose acetone antigen preserved a good potency at ph 6.4 as far as tested. From the practical point of view, it was decided to employ acetone ether antigen Table 2. Comparison of various techniques for the preparation of hemagglutinating antigen with infected suckling mouse brains * Acetone ether extracted antigen. ** Acetone ether extracted antigen additionally treated with protamine sulphate. *** Sucrose acetone antigen. **** Sucrose acetone antigen additionally treated with protamine sulphate. * Supplied from Ace Scientific Co., New Jersey, U.S.A.

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5 1961 NEGISHI VIRUS 55 for the complement fixation and acetone ether protamine antigen for HI test. Both antigens were stored in a freeze-dried state and when used reconstituted to a 25% preparation. Hemag-glutination Inhibition and Complement Fixation Tests The preliminary grouping of the virus was carried out with 7 mouse hyperimmune antisera against 16 units of the Negishi HAnin. Except a slight inhibition (1: 20) caused by JaGAr#21* serum, no inhibition was observed with 2 of Group A serum (AMM 2021, AMM 2354), yellow-fever 17 D serum, two from Bunyamwera group (Cachevalley, AMM 2222) or with Akabane serum. These results apparently indicated that the Negishi was a member of Group B arbor viruses. In the second step of serological approaches, the Negishi HAnin was tested against 15 sera from Group B together with 9 other antigens of Group B. In Tables 3 and 4 are presented reciprocals of serum dilution which gave a complete inhibition against 8 units of antigen with certain information of each antigen and antiserum. It has already been demonstrated by Casals (1957) that there are heavy immunological overlaps among certain members of Group B especially when hyperimmune sera are employed. The above picture of the HI test proved that this was actually the case. Though difference of one dilution should be considered as lying within the technical error range, it was observed in more than single occasion that some of the sera showed higher HI titers against heterologous antigens than against the homologous one. To make this complicated situation simpler, the complement fixation test was thought to be pertinent for subgrouping before, analysing the above results of HI in detail. Therefore, the following 12 combinations of antigens and antisera were subjected to a regular two-dimensional CFT. Because of the limitation in the instruments and in the time required for simultaneous performance of the tests, the whole test was divided into 3 sets so that each set included satisfactory controls with a hemologous system. As far as these tests are concerned, the results proved quite a high reproducibility with 1.7 to 2.2 units of complement. Fig. 1 is a summarized diagram which provided a definite proof that the Negishi belongs to the RSSE subgroup but never falls into the Japanese B-West Nile complex. In analysing the above results of CFT, it became clear that the Negishi was related to but remained distinct from Powassan. The slight difference observed between RSSE and Negishi did not seem to give a convincing proof for distinct entities of these viruses, however an appreciable degree of complement fixation found between Negishi serum and SLE antigen indicated a difference of Negishi from the other two viruses. From this point of view, it is of interest to recall the features of the virus in HI. In Table 3, where the initial sera were employed, the Negishi serum inhibited the homologous antigen in 1 : 80, RSSE antigen to a titer lower by two dilutions, and finally failed to inhibit Powassan antigen, whereas Powassan serum showed a titer higher by 3 dilutions against the homologous than against Negishi antigen. Furthermore, these relationships were practically the same even with the hyperimmune sera as can be seen in Table 4. Though the initial RSSE serum was able to demonstrate merely a difference of one dilution, it was rather extended with hyperimmune RSSE serum. It may be stressed here in Table 4 that the Negishi 5-injection serum inhibited the * One of the Japanese B strains isolated from mosquitoes in Gumma Prefecture in 1959.

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7 1961 NEGISHI VIRUS 57 Fig. 1. Immunological overlaps of Negishi virus among the representative members of Group B arbor viruses* in complement fixation test. * Naka: Japanese B strain Nakayama G-1: Japanese B strain G-one in the late level of passage JaGAr: Japanese B strain JaGAr#01 W N: West Nile strain Egypt 101 Powas: Powassan virus Den 1: Dengue Type 1 Hawaii YF: Yellow Fever 17 D strain ** Absissa and ordinate of each square denote dilutions of antiserum and antigen respectively; the upper-left corner representing 1: 4 and the lower-right, 1: 512 dilution. Less than 25% fixation was given by the shadow in each square. antigens of MVE and of West Nile in higher dilutions than the homologous. This would mean a biological complexity of HI tests rather than an immunological differentiation such as a relatively poor capacity of Negishi virus to yield HAnin. Conclusively, the Negishi virus belongs to RSSE subgroup and it is an entity distinct from Powassan virus. It is immunologically slightly different from RSSE.

8 58 OKUNO et al. Vol. 14 DISCUSSION The problem of whether the tick-borne arbor virus exists in Japan has always been one of the foci of discussion since a decade ago. It should be emphasized that when the isolation was made by Ando, there was not a single tick-borne virus in his laboratories. Besides, it was observed by Ando with a relatively early level of passage of the virus that the virus did not react serologically with Japanese B but did with SLE (Honda, 1951). This observation is in a close agreement with the results of our present study in CF, and presumably means that the virus was completely apart from Japanese B even in the early level of passage. There is, of course, a question as to whether this virus came from the patient materials or from the mice used for the original isolation work. In any event, however, it is a fact that a virus was isolated in this country which belongs to the tick-borne arbor virus group. Although it has not been possible to test systematically the specific HI antibodies of human residents in Kanto Plain, Japan, limited data at hand (approximately 200 samples) indicate an extremely small possibility of contamination of the vast majority of the people in Kanto Plain neither with the Negishi nor with RSSE. A special consideration should be made about the socio-ecological situation of Japan during the 1940 decade. It may admissibly be understood that this country was dealing with the huge population dynamics throughout the south-eastern and northern countries of Asia. There is no reason to exclude the possibility of an introduction of some virus such as tick-borne TP21 of Malaya at that time. In fact an epidemic of dengue Type 1 occurring during in the western part of Japan was virtually the case. Reasonably, it may be interpreted that there was incidentally a suitable environment for the dissemination of dengue virus while, in contrast to this, Negishi virus once appearing here did not find circumstances in which to cause more than a temporary sporadic epidemic. The relationships in HI between this virus and dengue, or the remainder of RSSE subgroup viruses such as KFD, TP21, BMF, OHF and swedish tick borne viruses has been at the moment left to be answered. SUMMARY Negishi virus, isolated by Ando from a fatal case of human encephalitis in Tokyo, Japan, in 1948 was subjected to identification studies with recently developed techniques of hemagglutination inhibition and complement fixation tests. It turned out to be a member of RSSE family among Group B arbor viruses. It is a distinct entity at least from RSSE or Powassan virus. The possibility of existence of the Russian-tick-borne virus in Japan was discussed. The authors are grateful to Drs. Ichiro Kobayashi, Takayuki Ogata for their collaboration, and the staffs of the Monday Discussion Meeting in the Department of Virology & Rickettsiology of our Institute for valuable advices. REFERENCES ANDO, K., KURATSUKA, K., ARIMA, S., HIRONAKA, N., HONDA, Y. & ISHII K.(1952): Studies on the viruses isolated during epidemic of Japanese B encephalitis in 1948 in Tokyo area. Kitasato Arch. Exper. Med., 24,

9 1961 NEGISHI V1R US 59 CASALS, J.(1957): The arthropod-borne group of animal viruses. Trans. N. Y. Acad. Sc., Ser. II, 19, CASALS, J.(1960): Antigenic relationship between Powassan and Russian Spring-Summer ence phalitis viruses. Canad. M. A. J., 82, CLARKE, D. H. & CASALS, J.(1958): Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. Am. J. Trop. Med. & Hyg., 7, FULTON, F. & DUMBELL, K. R.(1949): The serological comparison of strains of influenza virus. J. Gen. Microbiol., 3, HONDA, Y.(1951): Comparison of the immunological characters of a new virus gnegishi strain h and Russian spring-summer encephalitis virus. Kitasao Jikken Igaku, 24, ( text in Japanese). LOGRIPPO, G. A.(1958): Antigenicity of combined Ĉ-propiolactone and ultraviolet inactivated virus vaccines. J. Immunol., 80, LOGRIPPO, G. A.(1960): Investigations of the use of beta-propiolactone in virus inactivation. Ann. N. Y. Acad. Sci., 83, MCLEAN, D. M. & DONOHUE, W. L.(1959): Canad. M. A. J., 80, 708. MATSUYAMA, T., OYA, A., OGATA, T., KOBAYASHI, I., NAKAMURA, T. & KITAOKA, M. (1960): Isolation of arbor viruses from mosquitoes collected at live-stock pens in Gumma Prefecture in Jap. J. M. Sc. & Biol., 13, THEILER, M.(1957): Action of sodium desoxcholate on arthropod-borne viruses. Proc. Soc. Exper. Biol., 96, WARREN, J., WEIL, M. L., RUSS, S. B. & JEFFRIES, H.(1949): Purification of certain visuses by use of protamine sulphate. Proc. Soc. Exper. Biol. & Med., 72, ADDENDUM After this paper was submitted, staff doctors of RFVL have demonstrated the definite evi dences which indicated the distinct entity of Negishi virus from the known members of Russiantick-borne family viruses (Progress Report from RFVL, February 1961) These studies were characterized by the use not only of a regular serological procedure but also of the agar gel precipitation and antibody absorption techniques.