INTRABULBAR INOCULATION OF JAPANESE ENCEPHALITIS VIRUS TO MICE

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1 THE KURUME MEDICAL JOURNAL Vol. 15, No. 1, 1968 INTRABULBAR INOCULATION OF JAPANESE ENCEPHALITIS VIRUS TO MICE TOSHINORI TSUCHIYA Department of Microbiology, and Department of Ophthalmology, Kurume University School of Medicine, Kurume, Japan (Received for publication March 8, 1968) In the animal experiments, arboviruses are usually inoculated intracerebrally to susceptible animals such as mice. However, other inoculation routes, for instance, intranasal or intraperitoneal inoculation are sometimes used depend upon the experimental purposes. Although it is obvious that the virus inoculated by intraperitoneal or intranasal routes finally must reach the nerves of brains, it is not yet clear whether the virus goes directly to the central nerveous system or not. In the present paper, an intrabulbar inoculation of arbovirus which has not been used, so far, is described and the results obtained are compared to the data of routine inoculation route of arboviruses. Virus used MATERIALS AND METHODS Brains of 3-week-old mice infected with the Nakayama strain of Japanese encephalitis viurs (M-130 and 131) (JEV) were employed as a source of virus used. Inoculation procedures Each 0.05 ml of diluted virus materials were inoculated conjunctivally and intranasally, 0.02 ml of materials were used for both retrobulbar and intracerebral inoculations, and 0.01 ml of material was used for intrabulbar inoculation. Mice were treated under ether anesthetic when inoculated. Infectivity titration by animal experiments Three-week-old mice were used for infectivity titration of the virus ml of serial 10-fold diluted sample was inoculated intracerebrally into mice. Fifty per cent lethal dosis (LD50) was calculated according to Reed and Muench. Infectivity titration by tissue culture Chick embryo cell monolayers were prepared by seeding each bottle with 43

2 44 TOSHINORI TSUCHIYA 4 ml of cells obtained by trypsinization of 9-day-old chick embryos. The cells were suspended in 0.5 % lactalbumin hydrolysate, 10% inactivated bovine serum and % phenol red in Hanks balanced salt solution (BSS) with antibiotics., After overnight incubation at 37 Ž, the cultures were ready for use. Virus infectivities were assayed according to the plaque methods of Porterf ield and Kanda Inoue et al., with the following exception. The cell monolayers were washed with phosphate buffered saline (PBS) at ph 7.4, prior to seeding 0.2 ml of virus to each bottle. Following incubation at room temperature for the appropriate time, the infected cultures, nonwashed, were overlaid with 3.0 % agar mixed with Hanks solution containing 0.4 % lactalbumin hydrolysate, 0.3 % tryptose, 0.3 % bovine serum albumin V fraction, neutral red, NaHCO3, and antibiotics. Plaques were counted after incubation for 3 or 4 days at 36 Ž. Measurement of virus pathogenecity Virus pathogenecity was calculated by Gard's method : this is represented by the relationship between survival time and numbers of mice used in each virus dilution. RESULTS Effects of inoculation routes of JEV on infectivity to mice Infectivity titers of JEV inoculated by different routes to mice are demonstrated in Tables 1 to 3. In the tables, it was indicated that the virus could be-sent to the brains even by conjunctival and intrabulbar, as well as retrobulbar inoculation. Among the three inoculation methods : counjuctival, intrabulbar and retrobulbar routes, intrabulbar method always indicated the highest titer of infectivity, and also this was higher than intranasal inoculation method all the time. Furthermore, the virus titers obtained by intrabulbar inoculation were almost definite even if the virus titers measured by intracerebral inoculation were unsettled because of different sources of virus used. In addition, when pathogenicity grades of JEV to mice by different inoculation routes were calculated, from Table 3, according to Gard's method, it is obvious as shown in Fig. 1, that intrabulbar inoculation indicated the highest infectivity of JEV to mice, and that the lowest infectivity was demonstrated by intranasal inoculation. Viruses in noninoculated eyeball after intrabulbar inoculation Following intrabulbar inoculation of 0.01 ml of JE virus suspension (undiluted=10-1) to right eye, the left eye was removed on 1, 3, 5, 7 and 9 days, and it was homogenized in 0.5 ml of sterile saline containing streptomycin and penicillin, and virus titers in each eye were measured individually by plaque assay method in tissue culture. The results obtained are shown in Table 4.

3 INTRABULBAR INOCULATION OF JEV 45 TABLE 1 Effects o f inoculation routes of Japanese encephalitis virus on mortality of mice * survival days after inoculation S=survive TABLE 2 Effects of inoculation routes of Japanese encephalitis virus on mortality of mice TABLE 3 Effects of inoculation routes of Japanese encephalitis virus on mortality of mice

4 46 TOSHINORI TSUCHIYA Fig. 1. Comparisons of measurement of virus activities by different inoculation routes. TABLE 4 Virus in left eye after virus inoculation to right eye It was indicated that there was no evidence that the virus multiplied in the eyeball but each was maintained at the inoculation site during 1 to 9 days after inoculation. It was interesting also that the virus was detected in left eyeball from the beginning of inoculation to 9 days after inoculation. Virus growth modifications in brains of intrabulbar and intranasal inoculations by retrobulbar injection of immune serum In this section, two groups of experimental design were attempted. As control group, mouse brains were removed 1, 3, 5 and 7 days after intrabulbar or intranasal inoculation of undiluted virus (10-1) and each brain was homogenated in 3 ml of sterile saline containing streptomycin and penicillin. The virus titers

5 INTRABULBAR INOCULATION OF JEV 47 TABLE 5 Virus growth modifications in brains of intrabulbar and intranasal inoculations by homologous immune serum injection to retrobulbar Fig. 2. Virus multiplications in brains after intranasal and intrabulbar inoculation. Fig. 3. Virus growth modification in brain inoculated intranasally by retrobulbar injection of immune serum. in the brains were measured by plaque assay method. As experimental group, mice were treated with retrobulbar injection of 0.02 ml hyperimmune serum (neuralization titer =1: 2560) at the same time as intrabulbar or intranasal inoculation of virus suspension, and the brains were removed and the virus titers in them were measured by the same method as control group. The results obtained are shown in Table 5, Fig. 2 and 3. In the case of intrabulbar inoculation, the virus started to increase in the brain 3 days after inoculation and the titers gradually increased in the following days. In the experimental group ; immune serum injection to retrobulbar site following intrabulbar inoculation of virus, also the virus multiplied in the

6 48 TOSHINORI TSUCHIYA brain 3 days after inoculation and gradually increased. There was no difference between developing time of the virus in the brains of control and experimental groups, but the virus titers of experimental group were rather higher than that of control group. In the case of intranasal inoculation, the virus in control group started to increase 3 days after inoculation, as in the case of intrabulbar inoculation, but the virus multiplication in the brain was slightly inhibited by retrobulbar injection of immune serum. It might be thought, therefore, that immune serum neutralized some viruses through intranasal route at the retrobulbar site on the way to the brain. DISCUSSION There are no reports about experimental infection of arbovirus to mice by intrabulbar inoculation route. In general, arboviruses are inoculated experimentally to mice by intracerebral route, rarely by intranasal and intraperitoneal routes, but in the cases of latter, pathogenesis of the virus strongly reduce and survival times of mice extremely prolong, comparing to the case of intracerebral route. These findings represent that a large amounts of inoculated virus particles are inactivated by natural or nonspecific inhibitors contained in normal mice, on the course of virus attachment to susceptible cells. As non-specific inhibitors, nonantibody serum inhibitor, such as a or Francis inhibitor, Ĉ or Chu inhibitor, ć-inhibitor and others, ascitic fluids, and saliva, had been described. If JE virus is inoculated subcutaneously to mice, most of mice or all survive without any symptom. It indicates that inoculated virus is completely inactivated by normal body fluid containing nonspecific inhibitors. Then, intrabulbar and retrobulbar inoculations were attempted, because eyeballs are the most closed organ to the brain which is susceptible tissue to arbovirus. In other words, there is a very few opportunities that the inoculated virus is exposed to inhibitors. The results obtained here illustrate indeed that intrabulbar inoculation route has few obstacles to the inoculated virus arriving to the brain, because this method has the highest titer of virus infectivity among the methods of intranasal, conjunctival and retrobulbar inoculations. However, it is still obscure whether inoculated virus goes to the brain directly or indirectly. It is presumed that there are two opportunities which the virus goes to a brain; one opportunity is that the virus goes directly to the brain through the optic nerve ; this is supported by the experiments using Indian ink. Another opportunity is that the virus goes to the brain through blood stream after leaking out from a eyeball. Furthermore, it could be thought from the results shown in Table 4 that the virus which leaked out from inoculated eyeball might go to another eyeball.

7 INTRABULBAR INOCULATION OF JEV 49 There are some reports supporting that a virus causes a sympathetic ophthalmia. The results shown in Table 4, therefore, would give some informations about this speculation. Finally, on the course of these experiments, the pathway of virus to the brain from inoculation site by intranasal route was studied. In order to perform this experiment, hyperimmune serum was injected into retrobulbar site after intranasal inoculation of virus. If the virus particles after inoculated intranasally go through blood stream sited closely eyeball to the brain, the amounts of virus which arrive at the brain must be reduced by retrobulbar injection of hyperimmune serum. This speculation could be supported by the results indicated in Table 5. However, more precise experiments should be needed, in the future, in order to decide this hypothesis. SUMMARY Intrabulbar inoculation of Japanese encephalitis virus to mice was described. Virus multiplication in the brains inoculated by this method demonstrated definite titers, but these titers were lower than that of intracerebral inoculation. However, pathogenicity of the virus which was represented by survival time was higher than that obtained by intracerebral inoculation. Retrobulbar injection of immune serum slightly reduced the virus titers in the brains introduced by intranasal inoculation, but it had no effect in the case of intrabulbar inoculation. ACKNOWLEDGMENT The author wishes to express his appreciation to Prof. Masahiro Nakamura, Department of Microbiology and Prof. Yoshiya Masuda, Department of Ophthalmology for their helpful suggestions throughout the course of this work and in reviewing the manuscript. REFERENCES 1) REED, L. J. and MUENCH, H. A Simple method of estimating fifty per cent endpoints. Am. J. Hyg., 27, , ) PORTERFIELD, J. S. : Plaque production with yellow fever and related arthropod- orne viruses. Nature, 183, , ) KANDA INOUE, Y., IWASAKI, T. and KATO, H.: Studies of Japanese B encephalitis virus 1. Characteristics of mouse adapted and hamster kidney cell adapted Japanese B encephalitis viruses on plaque assay. J. Immunol. 87, , ) CARD, S.: Encephalomyelitis of mice II. A method for the measurement of virus activity. J. Exper. Med., 72, 69-77, ) FRANCIS, T., JR. : Dissociation of hemagglutinating and antibody measuring capacities of influenza virus. J. Exper. Med., 85, 1-7, 1947.

8 50 TOSHINORI TSUCHIYA 6) CHU, C. M. : The action of normal mouse serum on influenza virus. J. gen. Microbiol., 5, , ) COHEN, A., NEWLAND, S. E. and BIDDLE, F.: Inhibition of influenza virus hemagglutination : A difference of behavior in sera from a single species. Virology, 20, , ) GINSBERG, H. S. and HORSFALL, F. L., JR. : A labile component of normal serum which combines with various viruses. Neutralization of infectivity and inhibition of hemagglutination by the component. J. Exper. Med., 90, , ) CASSEL, W. A. : Vaccinia virus hemagglutinin inhibitor in ascitic fluids. Virology, 7, , ) FRANCIS, T., JR. and MINUSE, E.: Influence of saliva upon hemagglutination by influenza virus. Proc. Soc. Exp. Biol. and Med., 69, , ) IKUI, H., KIMURA, K. and IWAKI, 5.: Electron microscopic study of ultrathin sections of sympathetic ophthalmia. Acta. Soc. Ophthalm. Jap., 61, , ) IKUI, H., KIMURA, K. and NISHIO, T.: Further studies on sympathetic ophthalmia with electron microscope. Acta Soc. Ophthalm. Jap., 62, , 1958.