Florence S. Lief

Studies on the Soluble Antigen of Influenza Virus. I. The Release of S Antigen from Elementary Bodies by Treatment with Ether.

Soluble (S) antigen is readily released from influenza virus particles by exposure to ether. In order to obtain reproducible results with maximal yields of S and minimal damage to or loss of hemagglutinating and virus (V) antigen activities the technique described by Hoyle was modified and standardized. Elementary body suspensions which contained at least 5120 HA units per milliliter, and which failed to react per se with anti-S, were exposed to 12 volume of anesthetic ether at room temperature for periods up to 2 hours under constant agitation by a magnetic stirrer. Under these conditions, the hemagglutinating activity, as measured with guinea pig red cells, increased up to sixteenfold with all four influenza A strains tested; agglutination of chicken red cells was slightly enhanced with one of the strains and reduced twofold or more with the other three. The V antigen levels remained essentially unaltered. On the average, about 40 HA units of standard elementary bodies were required to yield one unit of S. Release of S became apparent within a few minutes of treatment. This was accompanied by nearly complete loss of infectivity and the sedimentation of the HA activity by high-speed centrifugation was markedly reduced. These results do not necessarily imply that the virus particles are disintegrated, as has been discussed.

Studies on the Soluble Antigen of Influenza Virus. III. The Decreased Incorporation of S. Antigen into Elementary Bodies of Increasing Incompleteness.

Abstract A comparison was made of the extent of incorporation of S antigen into fully infectious and partially incomplete elementary bodies (EB) as derived from allantoic fluids following inoculation of diluted standard virus on the one hand, and of undiluted native or heated (37°) standard, or undiluted passage seeds, on the other. Whereas on the average, 40 HA units (chicken red cells) of fully infectious EB ( ID 50 HA > 10 6 ) were required to yield one unit of S antigen after exposure to ether, increasingly more HA units were needed as the ID 50 HA ratios of the progenies decreased; i.e., for each successive tenfold reduction in the ID 50 HA ratio the number of HA units required increased in twofold steps. EB preparations derived from infected allantoic membranes yielded regularly less S antigen than those obtained from the allantoic fluids of the corresponding chick embryos in accordance with the fact that the tissue-bound virus materials always reveal ID 50 HA ratios 8–15 log 10 units lower than the progenies liberated from the cells. Furthermore, ether was found to be more detrimental to the hemagglutinating and antigen activities of EB suspensions derived from tissues than of those found in the allantoic fluids, suggesting that the former are as yet structurally deficient. The decreasing incorporation of S antigen into incomplete virus particles cannot be ascribed to a lack in its production in the tissues, since similar quantities were found free in the infected membranes as long as the total yield of hemagglutinins was not reduced. Exposure of incomplete virus derived from infected HeLa cell cultures to ether gave results similar to those observed with EB suspensions obtained from allantoic membranes. The implications of these findings have been discussed.

Studies on the soluble antigen of influenza virus. II. A comparison of the effects of sonic vibration and ether treatment of elementary bodies.

A comparison was made of the effects of sonic vibration and of exposure to ether on the release of S antigen from influenza virus. Elementary body suspensions (EB) obtained by high-speed centrifugation or by one cycle of adsorption onto and elution from red cells frequently reacted directly with anti-S sera. The presence of such “external” S depended upon the length of in ovo incubation and upon the strain of virus employed. Following sonic vibration the S titers increased in the EB preparations which initially revealed some S activity, and antigen became detectable in those originally free of measurable quantities. After sonic vibration or even only after second adsorption-elution cycles, the EB suspensions, as a rule, failed to react with anti-S. Although the data indicate that sonic vibration liberated some internal S, its release was not immediately accompanied by detectable losses in infectivity nor was a change noted in the degree of sedimentation of the virus particles by high-speed centrifugation. In any event, exposure to ether proved to be a more potent means of releasing internal S than sonic vibration since no additional S was liberated from ether-treated virus by the latter technique, whereas, in contrast, sonically vibrated EB suspensions on treatment with ether yielded apparently as much S as untreated virus particles. The implications of these results have been discussed.

EXPERIMENTAL PANENCEPHALITIS INDUCED IN SUCKLING MICE BY PARAINFLUENZA TYPE 1 (6/94) VIRUS: II. VIROLOGIC STUDIES

6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue, produced a chronic panencephalitis when inoculated intracerebrally into suckling ICR mice. Immunofluorescent staining revealed 6/94 viral antigen in ependyma, meninges, choroid plexus, and perivascular parenchy-mal sites from day 3 to 128 days after infection. Hemadsorption-neutralizing antibody was first detected between 20–25 days after infection and remained at high titers for 7 months. Using embryonated chicken eggs, virus was recovered from mouse brains for only 8 days, but could be recovered from brains grown in vitro as explants for 37 days after infection. In cell lines established from explanted brain tissue, immunofluorescence was the most sensitive indicator of virus presence, although infectious virus was not produced. Fusion of these mouse brain cells with human (WI38) indicator cells was the most effective means of rescuing 6/94 virus.

Antigenic Variation among Parainfluenza Type 1 (Sendai) Viruses: Analysis of 6/94 Virus

6/94 virus, isolated originally from a multiple sclerosis (MS) patient, was compared antigenically with related parainfluenza type 1 strains. These included two Sendai strains of mouse and two Sendai strains of reported human origin as well as the HA2 strain. By standard hemagglutination inhibition (HI) or hemadsorption neutralization (HAD-N) tests or by the complement-fixation (CF) cross-block titration test for detecting surface antigens, 6/94 virus and the Sendai virus strains were indistinguishable from each other but distinct from the HA2 strain. By the kinetic HI test, however, 6/94 virus could be readily differentiated from the Sendai viruses isolated from mice and more closely resembled those recovered from man.

Studies on the soluble antigen of influenza virus: IV. Fractionation of elementary bodies labeled with radioactive phosphorus☆

Abstract A comparison was made of the extent of incorporation of radioactive phosphorus into infectious and incomplete influenza virus particles. It was found that regardless of the ID 50 HA ratios of the progenies, the CPM HA ratios were of the same order; i.e., noninfectious hemagglutinins were as radioactive as infectious virus. Following treatment of labeled standard elementary bodies (EB) with ether, 15 to 20% of the isotope appeared in the ether phase (EE). On separating the aqueous phase (EEB) into HA and S antigen fractions, it was noted that most of the remaining P32 was associated with the latter. Chemical fractionation of labeled standard EB suspensions by a modification of the Schmitt-Thannhauser method confirmed previous reports that the isotope was mainly in the phospholipid (hot alcohol soluble) and in the nucleic acid (hot TCA soluble) fractions. The EEB preparations revealed a relative increase of radioactivity in the cold TCA fraction and decreases in the alcohol and hot TCA extracts. Thus exposure to ether resulted in the breakdown of some viral constituents. Most of the residual phospholipids (not removed by ether treatment) were found to be associated with the HA particles, whereas the labeled components soluble in hot TCA were largely recovered in the S fractions. On chemical fractionation of labeled incomplete virus preparations, it was noted that the relative concentrations of the isotope in the hot TCA extracts decreased with the reduction of the ID 50 HA ratios of the progenies. Corresponding increases were noted in the cold TCA fractions. However, the ratios between the CPM values of the hot TCA extracts and the S antigen units present in the virus were found to be of the same order regardless of the composition of the progenies studied. Various difficulties were encountered in these studies, which are discussed together with the significance of the results and their relation to previous biological, chemical, and physical observations.

I-cell disease: intracellular desialylation of lysosomal enzymes using an influenza virus vector.

It has been proposed that I-cell disease results from a primary deficiency of acid neuraminidase activity. Infection by influenza virus of fibroblasts from a patient with I-cell disease resulted in the production of abundant intracellular alpha2-3 neuraminidase activity. Despite electrophoretic evidence of desialylation of intracellular and fibroblast-secreted arylsulfatase (EC 3.1.6.1) and beta-hexosaminidase (EC 3.2.1.30) from the infected cells, there was no consequent alteration of the abnormal distribution of beta-hexosaminidase activity between the intracellular spaces characteristic of I-cell disease. This suggests that deficiency of alpha2,3 neuraminidase activity is not the primary biochemical defect in I-cell disease.