Ten rabbits were given a primary course of immunization by 4 weekly intravenous injections of herpes virus, and 1.5 years later 3 of them showing low titers of neutralizing antibody received 1 booster injection. The serological response in the primary immunization was characterized by an early development of complement-requiring neutralizing (CRN) antibody ahead of that of non-complement-requiring neutralizing (N) antibody, the CRN/N ratio being 8 to 128 within 3 weeks. In contrast, N antibody appeared much faster after the booster immunization, the CRN/N ratio approaching 4 within 1 week. The early type IgG, whose neutralizing activity was enhanced by complement (C') about 16-fold, was distinct from the late type IgG which could not be enhanced by C' more than 4-fold. The late type IgG appeared after 4 weeks and 3 days in the primary and booster immunizations, respectively. Serological examinations of human patients suggested the occurrence of the booster type response in the case of repeated infections among adults.Earlier reports from our laboratory pointed out that rabbits [21,22] and guinea pigs [23] immunized with herpes simplex virus developed early neutralizing antibody which could be detected only when complement (C') was present in the in vitro neutralization system. This new type of antibody was designated C'-requiring neutralizing (CRN) antibody. The appearance of CRN antibody preceded that of the classical neutralizing (N) antibody in such a way that early sera often showed an all-or-none type neutralization reaction depending upon the presence or absence of C'. Similar phenomena were found with vaccinia [8], bovine parainfluenza [6] and simian herpes SA-8 viruses [11].In the case of herpetic infections of humans, too, it was frequently observed that CRN antibody appeared at high titers before the development of N antibody, so that the detection of CRN antibody served for an early diagnosis of the diseases [23]. In contrast, Heineman [5] could not find any difference between the early and convalescent sera of adult patients in the enhancement of neutralization by C'. When Lerner et al. [7] examined patients of herpetic encephalitis older than 10 years, half of them demonstrated CRN/N ratios of 4 or more in the early stage.This discrepancy of data seemed to indicate different serological responses between

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It has been established that rabbits and guinea pigs immunized or infected with herpes simplex virus develop early neutralizing antibody which functions only in the presence of complement (C') [9,[40][41][42]. This antibody was named C'-requiring neutralizing (CRN) antibody [40]. Herpetic infections of man also stimulate the development of CRN antibody in advance of ordinary neutralizing (N) antibody, so that the detection of CRN antibody in an early serum sample can give a positive diagnosis [42], although this means of early diagnosis decreases in practicability in the case of repeated infections among adults [12,20] [36,37] and simian herpes SA 8 viruses [29] have revealed occurrence of similar phenomena. However, the data presented have not necessarily coincided as to the extent to which the neutralizing activity of early serum is enhanced by C' as compared with that of late serum. In the case of herpes virus, early CRN antibody frequently far exceeds N antibody in level and, as a consequence, enhancement by C' of the neutralizing activity of such a serum is almost of the all-or-none type. A similar fact has been noted with vaccinia [23], bovine parainfluenza [17] and simian her-

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SummaryThe faint plaques formed with HEP Flury strain of rabies virus in chick embryo cells by the original method were thought ascribable to acid production from virus-infected cells coupled by the weak buffering action of the agar overlay medium. When the overlay medium contained appropriate concentrations of alkalies, clear plaques could be observed. The optimal conditions were (i) incorporation of 0.02% ~TaOH, 0.0025 ~ Tris-HC1 buffer of pI-I 8.2 and 2% calf serum in the base overlay medium, and (if) neutral red staining after 7 days' incubation at 35~ Under these conditions the plaque size was approxilnate]y 2 mm in diameter and autointerference caused by higher mnltip]icity of infection showed a diminishing trend. Mouse passaged NOPM strain also formed fairly clear plaques when agarose was substituted for agar, the plaque titer being almost equal to its mouse LD titer. That these plaques were formed by infection of the virus was testified by a neutralization test using a standard antiserum prepared with rabbit passaged CVS strain. Adsorption of rabies virus to chick embryo cells was found to proceed slowly, the maximal adsorption requiring 4 hours or longer. The adsorption efficiency was equM between 35 ~ and 37~