Kamesaburo Yoshino

Studies on the neutralization of herpes simplex virus: I. Appearance of neutralizing antibodies having different grades of complement requirement☆

Abstract When rabbits were immunized with herpes virus, the production of early complement-requiring neutralizing antibody was followed by the appearance of neutralizing antibodies with a lower requirement for complement, which could be detected either by testing in the presence of lower concentrations of complement or by a prolonged incubation at 37° of virus-serum mixtures in the absence of complement. Kinetic experiments revealed that the virus-neutralizing velocity varied among sera of different immune stages when an equivalent concentration of antibody was tested, and that this velocity changed depending upon the amount of complement added, although the addition of complement did not influence the level of the persistent fraction. Neutralizing antibody in the γ-globulin fraction of an early serum and that of a hyperimmune serum both showed the properties characteristic for 7 S antibody, although a clear difference was seen in the requirement for complement.

THE APPEARANCE OF COMPLEMENT-REQUIRING NEUTRALIZING ANTIBODIES BY IMMUNIZATION AND INFECTION WITH HERPES SIMPLEX VIRUS.

Abstract Rabbits immunized with herpes simplex virus developed complement-requiring neutralizing antibodies at an early stage, usually reaching a peak titer before there was an appreciable rise of non-complement-requiring neutralizing antibodies. This was also true with rabbits that received corneal infection with herpes virus. Neutralization kinetics show a difference in virus-neutralizing velocity between the complement-requiring and nonrequiring antibodies. In tests with sera of persons possessing persistent antiherpes antibodies, enhancement of neutralizing antibody titers by the addition of complement was only approximately twofold on the average.

STUDIES ON THE NEUTRALIZATION OF HERPES SIMPLEX VIRUS. II. ANALYSIS OF COMPLEMENT AS THE ANTIBODY-POTENTIATING FACTOR.

Abstract The potentiating effect of unheated guinea pig serum upon the neutralizing antibody in early immune rabbit serum showed a parallelism with hemolytic units of complement contained therein, when several lots of guinea pig serum stored under different conditions were compared. Absorption of complement from guinea pig serum by a mixture of egg albumin and anti-egg albumin rabbit serum removed the antibody-potentiating capacity completely. By testing the effect of depletion of each component of complement from guinea pig serum, it was found that all the four components were necessary for the antibody-potentiating action.

Studies on the neutralization of herpes simplex virus: III. Mechanism of the antibody-potentiating action of complement

Abstract The mechanism of the antibody-potentiating action of complement was investigated. In the neutralization of herpes virus by immune serum, formation of virus-antibody complexes which are not necessarily neutral precedes viral inactivation. In the case of early immune serum containing complement-requiring neutralizing (CRN) antibody, the interval between virus-antibody binding and viral inactivation is very long, and the action of complement is to shorten this interval. The virus-CRN antibody binding, or sensitization of virus by CRN antibody, as well as the action of complement is quicker at 37° than at 4°. The virus-CRN antibody complex formed in the absence of complement is not easily dissociable by simple dilution, nor by other means such as sonic vibration; has no blocking effect against hyperimmune antibody; and remains sensitive to the antibody-potentiating action of complement during an early stage of adsorption to cells.

Autointerference of Rabies Virus in Chick Embryo Fibroblasts.

Summary When HEP Flury strain of rabies virus was inoculated onto chick embryo fibroblasts at a multiplicity of higher than 0.1, no plaques were formed and almost all cells remained stainable with neutral red. Monolayers pretreated with such a concentration of HEP Flury virus showed interference with plaque formation by vaccinia and WEE viruses. This was not due to presence of interferon in the seed virus suspension, nor to the presence of tissue components in the inoculum. In bottle cultures of chick embryo cells, virus growth after infection at higher multiplicities was accompanied by production of a considerable amount of interferon, and the highest virus yield was obtained when the multiplicity of infection was about 0.01.

A SIMPLE PATTERN METHOD FOR THE SERUM NEUTRALIZATION TEST WITH HERPES SIMPLEX VIRUS IN CHICK EMBRYO MONOLAYERS.

A simple method for the serum neutralization with herpes simplex virus was developed utilizing the ability of this virus to form plaques in chick embryo monolayers. A constant virus-varying serum system was adopted. The new device was the use of eight small agar cover slips (ACS), prepared by punching out of a solidified thin layer of an agar overlay solution, for inoculation of virus-serum mixtures onto a monolayer dish, so that each serum sample could be titrated on one monolayer dish. Reduction of virus infectivity was evidenced by changes in the pattern from confluent to negative or a small number of scattered plaques. When a certain dilution of seed virus was used constantly, a fairly high reproducibility of serum endpoints was demonstrated, which were comparable to those obtained by other routine neutralization methods. Serum samples from 39 normal healthy persons were tested by this method and by complement fixation tests, and the serum endpoints obtained by the two tests showed a good parallelism.A simple method for the serum neutralization with herpes simplex virus was developed utilizing the ability of this virus to form plaques in chick embryo monolayers. A constant virus-varying serum system was adopted. The new device was the use of eight small agar cover slips (ACS), prepared by punching out of a solidified thin layer of an agar overlay solution, for inoculation of virus-serum mixtures onto a monolayer dish, so that each serum sample could be titrated on one monolayer dish. Reduction of virus infectivity was evidenced by changes in the pattern from confluent to negative or a small number of scattered plaques. When a certain dilution of seed virus was used constantly, a fairly high reproducibility of serum endpoints was demonstrated, which were comparable to those obtained by other routine neutralization methods. Serum samples from 39 normal healthy persons were tested by this method and by complement fixation tests, and the serum endpoints obtained by the two tests showed a good parallelism.

Infection of the One-Day Old Fertile Hen's Egg with Rabies Virus. VI. Strain Differences in the Egg Adaptability.

Three street, three rabbit-fixed and three mouse-fixed strains of rabies virus were examined for growth rates, infective titers and embryo-cidal action in 1-day and 7-day old eggs, with the results that there were wide differences among these strains. In the case of egg-infective strains, 1-day eggs supported better viral growth and were more sensitive to virus infection than 7-day eggs. The mouse-fixed strains were generally of higher egg infectivity than the rabbit-fixed strains, but, as far as tested, serial passages in different host animals did not alter the egg infectivity of any so treated strain. A rabbit-fixed strain Nishigahara and a mouse-fixed strain Takamen were passaged serially in 1-day eggs, whereby the process of adaptation also reflected the strain differences in the egg infectivity between the parent strains. Adaptation of Nishigahara strain proceeded gradually at first with increasing viral yields and then, in much later passages, with intensified embryo-cidal action, whereas Takamen strain gave almost equally high yields throughout with increased embryo-cidal activity from a very early passage level. Furthermore, the ratio of mouse- and 1-day egg-infective titers of the 1-day egg-adapted virus differed between these two strains. Finally, a higher maximum virus yield was obtained in the infected eggs with the 1-day egg-adapted Nishigahara than with Takamen virus.Three street, three rabbit-fixed and three mouse-fixed strains of rabies virus were examined for growth rates, infective titers and embryo-cidal action in 1-day and 7-day old eggs, with the results that there were wide differences among these strains. In the case of egg-infective strains, 1-day eggs supported better viral growth and were more sensitive to virus infection than 7-day eggs. The mouse-fixed strains were generally of higher egg infectivity than the rabbit-fixed strains, but, as far as tested, serial passages in different host animals did not alter the egg infectivity of any so treated strain. A rabbit-fixed strain Nishigahara and a mouse-fixed strain Takamen were passaged serially in 1-day eggs, whereby the process of adaptation also reflected the strain differences in the egg infectivity between the parent strains. Adaptation of Nishigahara strain proceeded gradually at first with increasing viral yields and then, in much later passages, with intensified embryo-cidal action, whereas Takamen strain gave almost equally high yields throughout with increased embryo-cidal activity from a very early passage level. Furthermore, the ratio of mouse- and 1-day egg-infective titers of the 1-day egg-adapted virus differed between these two strains. Finally, a higher maximum virus yield was obtained in the infected eggs with the 1-day egg-adapted Nishigahara than with Takamen virus.

Studies on the neutralization of herpes simplex virus IV. Properties of virus sensitized with complement-requiring neutralizing (CRN) antibody at 37° and 0°☆

Abstract Sensitization of herpes virus by CRN antibody at 37° was more rapid than at 0°, reaching a final state within 1 hour in which less than 0.1% of the initial virus remained insensitive to the inactivating effect of complement. The virus sensitized at 37° was inactivated rapidly by addition of complement, whereas the virus sensitized at 0° showed a slower inactivation even with excessive complement, the velocity being progressively faster with increasingly longer sensitization. When grown in chick embryo cells, the 37°-sensitized virus showed a retardation of growth initiation by 2 hours; it was suggested that this retardation was not attributable wholly to slow penetration of the cell membrane. No such retardation could be observed in the case of the 0°-sensitized virus. It has been concluded from these data that the CRN antibody-sensitized state of virus is not uniform but there are different states from a low to a high sensitization, and that the highly sensitized virus behaves differently from native virus in the early stages of infection of cells.

Infection of the one-day old fertile hen's egg with rabies virus. VII. Comparison of viral yields obtained by inoculation of eggs of different ages with different strains and production of vaccine.

The mouse-fixed Takamen, moderately 1-day egg-adapted Nishigahara, 7-day egg passage HEP Flury strains of rabies virus and their highly 1-day egg-adapted sublines were inoculated into 1-day, 3-day, and 5-day eggs to examine the virus yields in the embryo. It was consistently observed that the 1-day egg infection could give the largest total amount, as well as the highest concentration of virus in the embryo. The moderately 1-day egg-adapted Nishigahara strain was found to be best fitted for the production of 1 -day egg vaccine, because the size of the embryos harvested was sufficiently large and the concentration of virus yielded therein was higher than could be obtained with other strains. The egg-infective properties of this particular subline of Nishigahara strain could be maintained by transferring this virus to mouse brain passage. Five lots of 1-day egg vaccine prepared with this strain by means of UV inactivation possessed Habel indices of more or less 1,000 LD. One of these lots showing the lowest Habel index turned out to have more than 7 times as high a potency as the U. S. reference vaccine when tested according to the U. S. Minimum Requirements.

Unusually Large-Plaque Mutant of Sindbis Virus

Summary When Sindbis virus plaques were formed in chick embryo fibroblasts (CEF) at 40°C, plaques varying in size were observed. Plaques measuring less than 2 mm (sp) were subjected to cloning. Out of 51 sp plaques so examined, three yielded predominantly, on passage through CEF, unusually large-plaque (u) mutants. This mutant as well as the parent large-plaque (lp) and sp viruses were purified by consecutive clonings. Mixed infection of CEF with the sp and u viruses tended to exclude the sp virus. Contrarily, mixed infection with the lp virus and the u mutant tended to exclude the latter. Thus for maintenance of the u mutant, frequent clonings had to be done, because otherwise an incidental appearance of the parent type virus by back mutation seemed to result in exlusion of the u mutant virus. The u mutant as well as the sp virus was more sensitive to interferon than the lp virus. The reason why the interferon-sensitive u mutant can grow more rapidly and yield a higher amount of virus than the parent lp virus remains to be elucidated.