Gordon C. Brown

Rubella Antibodies in Human Serum: Detection by the Indirect Fluorescent-Antibody Technique

Antibodies for rubella virus were detected in human serum and titrated by the indirect method of immunofluorescence; a chronically infected, continuous line of monkey kidney cells was used as antigen. Positive reactions were obtained with serums from convalescent patients or persons who had been exposed to the virus while serums from patients in the acute stage of the disease and those from unexposed individuals were negative

Specific Response of the Immunoglobulins to Rubella Infection

The teratogenic consequences of rubella infection early in pregnancy are well known. However, experience with the rubella epidemic in 1964 revealed unexpected virologic and serologic consequences of maternal infection. Despite the fact that the rubella syndrome was caused by maternal infection prior to the immunologic maturity of the fetus, serologic immunity rather than tolerance was observed in older children with the syndrome (1, 2). Furthermore, rubella virus was found to persist in the affected infants for many months despite the presence of serum antibodies (3-5). Attempts to equate the presence of rubella antibodies in these infants with an active immune response by means of conventional serology was complicated by the presence of passively acquired maternal antibodies of the IgG variety which persist for several months. Since IgA and IgM do not cross the placental barrier readily, the association of antibody activity within the IgM and/or IgA class of globulins of the infants serum would indicate active immunity. The following studies were carried out in order to characterize the classes of immunoglobulin which responded specifically to rubella infection and to determine their chronology. Observations were also made regarding the classes of rubella antibodies in sera from “normal” newborns and infants with congenital rubella. Materials and Methods. Rubella antibodies were demonstrated by the indirect fluorescent antibody method as described by Brown et al. (6). Whereas conventional methods of serum fractionation and antibody titration were cumbersome, this technique provided a rapid and reliable method for identification of the antigenic class of antibodies (IgG, IgA, or IgM) in whole serum which had combined with viral antigen. Cover slip cultures of a chronically infected line of monkey kidney cells (7) fixed in acetone were employed as an antigen source. The conjugated antisera used in this study were purchased from Hyland Laboratories as fluorescein conjugated antisera prepared in goats against human IgG, IgA, and IgM globulins, respectively. Before use these sera were fractionated by DEAE chromatography as described by Riggs et al. (8).

Effect of DL-ethionine on poliomyelitis virus growth in tissue culture.

Conclusions The Lansing strain of poliomyelitis virus requires L-methionine for its growth and multiplication in cultures of human embryonic brain tissue. Its growth can be materially inhibited with DL-ethionine, a structural analogue of the essential amino acid. The inhibitor acts by interfering with the biosynthesis of the poliomyelitis virus just as it suppresses the growth of influenza virus. This technic affords a procedure for studying the growth requirements of poliomyelitis virus and for testing materials which may influence its growth.

Differential and Specific Inhibition of ECHO Viruses by Plant Extracts.

Summary 1) Comparison of antiviral activity of several plant extracts against several ECHO viruses revealed that M2, prepared from a strain of Calvatia gigantea significantly inhibited only ECHO 4 and 11; while M4, a different preparation from the same strain, inhibited only ECHO 7 and 8; and M14, from a species of Cattleya, inhibited ECHO 2. The effectiveness of M2 in vitro was greatest when the cells were pretreated and when the virus inocula did not exceed 100 TCID50. 2) Specificity of the antiviral action of M2 was demonstrated by inhibition of one M2-susceptible virus followed in the same culture by growth of a second M2-nonsusceptible virus. Inhibition continued, however, if the cultures were reinoculated with a second M2-susceptible virus. 3) Activity of M2 was demonstrated in vivo by its ability to suppress the antigenicity of an M2-susceptible virus. 4) The results demonstrate strikingly the capacity of the materials to prevent infection of susceptible cells by specific viruses although the cells retain their physiologic competence to support growth of other viruses and the virus is not directly affected by the inhibitory material. Obviously the effect must be directed to modification of reactions essential to viral development but not injurious to functional integrity of the cell.

Application of direct and indirect immunofluorescence for identification of enteroviruses and titrating their antibodies.

Summary Tissue culture cells infected with various enteroviruses show specific immunofluorescence when stained directly with conjugated fractionated antiserum. The indirect method of fluorescent antibody determination is an even more satisfactory diagnostic technic since it makes possible the detection and titration of enterovirus antibodies actively acquired following clinical or subclinical infection or from vaccination.

Antiviral Action of a Mold Filtrate on Experimental Poliomyelitis in Gynomolgus Monkeys.

Summary The crude filtrate of a penicil-lium mold, M-8450, was shown to have antiviral action in cynomolgus monkeys inoculated subcutaneously with the Mahoney strain of Type 1 poliomyelitis. Treatment with at least 100 ml of this crude filtrate reduced the morbidity and increased the incubation period of infected animals. Less intensive treatment appeared to be less effective. The antiviral action of this substance appears to merit further study.

Effect of benzimidazole on experimental poliomyelitis in mice and monkeys.

Summary 1. The influence of benzimidazole on experimental poliomyelitis in mice and monkeys was investigated. The subcutaneous administration of 250 mg/kilo prolonged the incubation period of infection with the Lansing strain of Type 2 poliomyelitis in mice and reduced the mortality caused by minimal quantities of this virus. 2. Infection of adult mice with intracerebrally inoculated MEF-1 strain of Type 2 virus was not affected by this compound which even appeared to enhance infection in suckling mice inoculated intraperitoneally with this same strain. 3. In monkeys the incubation period of infection with the Mahoney strain of Type 1 virus was prolonged slightly by limited treatment with benzimidazole, but the mortality was not reduced. More intensive treatment was without effect. 4. The advisability of further work with this and structurally similar compounds is discussed.

Embryonic Chick Antigens for Complement Fixation with Viruses of Eastern and Western Equine Encephalomyelitis.

The complement-fixation test has been increasingly employed in the identification of virus infections of the central nervous system. With one exception all complement-fixing antigens thus far employed with equine encephalomyelitis have been prepared from animal brain tissue, usually that of the mouse. 1 - 7 Mohler, 8 using a formolized antigen prepared from chick embryos inoculated with the virus of Western equine encephalomyelitis, reported complement fixation with serum from convalescent or immunized horses. Because of its size a single infected chick embryo contains many times as much virus as an infected mouse brain and in view of the greatly decreased incubation period for maximal production of virus further investigation of this source of antigen was suggested. Equine encephalomyelitis antigen. A strain of Eastern virus obtained from Dr. A. B. Sabin and the McMillan strain of Western virus received from Dr. J. Casals were employed. The viruses had been maintained at high titer by continued intracerebral passage through mice. They were then subjected to 13 rapid passages in hens′ eggs containing 10-day-old embryos by inoculation of 0.1 cc of 1:100 suspension of infected embryonic tissue onto the chorioallantoic membrane. The embryos were removed after 18-24 hours and suspended in physiological salt solution to give a concentration of 20% by weight. The suspension was centrifuged at 2,500 r.p.m. for 15 minutes and the supernatant removed and stored at −70°C. Before use as antigen the virus suspension was thawed and centrifuged at 3,000 r.p.m. for 15 minutes. The clear supernatant was diluted with an equal volume of physiological salt solution. The preparations, still infectious for mice in titers of 10-8 with Eastern virus and 10-6 with Western virus, were then tested in order to determine their complement fixing ability in the presence of immune serum.

Effect of Fluoroacetate upon Poliomyelitis in Monkeys.

Summary 1. Cynomolgus monkeys were treated with 5 mg/kilo of sodium monofluoroacetate on the day of, or 3 days following, subcutaneous inoculation of Type 1 poliomyelitis virus. 2. Nine of 42 monkeys which received drug on the Third day of virus resisted clinical infection as did 5 of 23 animals receiving drug on the third day after virus. All control animals became paralyzed. 3. Citrate did not accumulate in brain tissue of drug-treated monkeys suggesting that the action of fluoroacetate was at some site other than the brain. 4. The significance of these findings is discussed.

Recovery of virus from throats of poliomyelitis patients.

Summary Virus was recovered from 4 of 7 throat swabs and from 3 of 9 throat washings of poliomyelitis patients collected during the first 3 days after onset of symptoms. In another series of 16 throat washings collected from 3 to 13 days after onset, virus was isolated from 2 patients 11 days after onset of disease.