Sadako Taniguchi

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.

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.

Demonstration of the Eclipse Phase of Herpes Simplex Virus by a New De-Embryonation Technique.

Abstract De-embryonated eggs were prepared by a special technique of agar filling so that the ectodermal side of the chorioallantoic membrane (CAM) was utilized for infection. When herpes virus was inoculated by placing a virus-smeared cover slip onto the CAM, approximately 40% of virus was adsorbed to the CAM cells within 2 hours and the viral growth was similar to that in the intact egg CAM previously studied. In the initial stage of infection, it was impossible to separate residual active virus from the cells completely even after fifty repeated washings with saline; nor was the virus completely neutralized by the addition of a potent antiserum. However, the residual virus was as sensitive to ultraviolet (UV) irradiation as was free virus and could be reduced to an undetectable level by a mild UV irradiation followed by ten washings. Most of the eggs so treated kept their virus-replicating potentials unchanged and demonstrated the eclipse phase directly. Newly formed virus seemed to appear within and outside infected cells 10–11 hours after inoculation.

Serum neutralization test with western equine encephalitis (WEE) and Japanese encephalitis (JE) viruses using the agar cover slip (ACS) inoculation technique

The conventional neutrMization test of viruses has been too cumbersome to be widely used for serodiagnosis, and a number of a t tempts have been done to simplify the procedure. As one of such a t tempts , Umeda et al. (1) worked out the agar cover slip (ACS) method for herpes simplex virus, in which virus.serum mixtures were inoculated to ceils under cover slips made of overlay medium, and endpoints were determined by reading plaque patterns appearing at the inoculation spots, taking negative or few scattered plaques as positive neutralization when the control spot showed confluent plaques. Advantages of this method, were tha t one monolayer dish was enough to t i t ra te each serum sample, and tha t reading of results was not time-consuming. The standard deviation of endpoints determined by this method was 10.67 log~ (2). When this method was applied to two arboviruses, W E E and JE , however, first tests failed to demonstrate clear endpoints, because virusserum mixtures contained comparatively high levels of unneutralized virus, especially in the neighborhood of the endpoint dilution of antiserum, and consequently inoculation of such mixtures under ACS did

Disparity between viral growth and cellular proliferation in the hen egg chorioallantois infected with a fresh isolate of herpes simplex virus

Abstract When a fresh isolate of herpes simplex virus was inoculated onto the chorioallantoic membrane (CAM) of 12-day eggs, the virus titer increased only during the first 2 days, and an abrupt fall took place between days 2 and 3 which was not due to viral spread to the other parts of the egg. Cellular proliferation progressed for some time even after this stage, continuing with a constant low titer of active virus. When the cells of the CAM were trypsin dispersed and cultured in vitro , a new increase of active virus was seen. However, no evidence was obtained for the presence of latent infection in the proliferating cells of the virus-infected CAM. An assay for interferon indicated its appearance in the virus-infected CAM from the 24th hour post-infection. The effect of the interferon was greater upon the fresh strain than upon egg-adapted strains. Eggs infected with the fresh strain showed resistance to superinfection with the homologous virus, but supported growth of the egg-adapted strains. The function of the cellular proliferation in localizing and suppressing viral infection is discussed.