The effect of polysaccharides on the reaction between erythrocytes and viruses,with particular reference to mumps virus

Polysaccharides which cause inhibition of the multiplication of mumps virus in the allantoic sac may or may not cause inhibition of hemagglutination by the virus. Moreover, such substances may or may not prevent adsorption of the virus by erythrocytes. The available evidence indicates that polysaccharides active as inhibitors do not block adsorption of mumps virus by cells of the living allantoic membrane. With influenza A, influenza B, and Newcastle disease viruses, as well as with PVM, there also appears to be a lack of correlation between the in vitro and in vivo inhibiting activity of polysaccharides.     

THE MODIFYING EFFECTS OF CERTAIN SUBSTANCES OF BACTERIAL ORIGIN ON THE COURSE OF INFECTION WITH PNEUMONIA VIRUS OF MICE (PVM)

Evidence is presented which indicates that certain polysaccharide preparations derived from various bacterial species, as well as similar materials not of bacterial origin, are capable of lessening the severity of infection with pneumonia virus of mice (PVM) and inhibiting multiplication of the virus in mouse lungs infected with this agent. It seems probable that modification with respect to the virus is mediated by a substance which may be polysaccharide in nature.     

SPECTRUM AND CHARACTERISTICS OF THE VIRUS INHIBITORY ACTION OF 2-(α-HYDROXYBENZYL)-BENZIMIDAZOLE

2-(α-Hydroxybenzyl)-benzimidazole (HBB) inhibited the cytopathic effects of the following enteroviruses: polio 1 to 3; Coxsackie A9; Coxsackie B 1 to 6; and ECHO virus types 1 to 9, 11 to 21, and 24 to 27. The following enteroviruses were not inhibited: Coxsackie A types 7, 11, 13, 16, and 18; and ECHO types 22, 23, and 28. Other HBB-insusceptible viruses were: arbor B and C, reo 1 to 3; adeno 2 to 4; influenza B; para-influenza 2 and 3; mumps; herpes simplex, and vaccinia. HBB had no inactivating effect on viral infectivity, but rather inhibited some intracellular step in the reproductive cycle of susceptible viruses. With all viruses examined, inhibition of viral cytopathic effects appeared to be due to inhibition of virus multiplication. Virus inhibition by HBB was demonstrable in monkey kidney, HeLa, and ERK cells. HBB-susceptible viruses varied quantitatively in their susceptibility to the compound, and different strains of the same virus also exhibited varying susceptibility. No relationship was found between attenuation of polioviruses and their susceptibility to the compound. After passage of HBB-susceptible enteroviruses in the presence of the compound, virus populations with lowered susceptibility to HBB were obtained. At virus inhibitory concentrations, HBB did not affect the morphology of cells, nor the following cellular metabolic activities: oxygen uptake; glucose utilization; lactic acid production; and incorporation of adenosine into RNA, and of alanine into proteins. The rates of multiplication of HeLa and ERK. cells were not significantly altered by HBB at virus inhibitory concentrations.     

Effects of Ribavirin on BHK-21 Cells Acutely or Persistently Infected With Mumps Virus

The effects of ribavirin on BHK-21 cells acutely infected with mumps virus were compared to the effects of the drug on the same cell line persistently infected with mumps virus. Visible cytotoxicity was minimal for both cell types; however, there was an inhibition of cell replication with increasing drug concentrations. Ribavirin had marked antiviral activity against both the acute and persistent infections as determined by an inhibition of hemadsorption plaque formation, decreased immunofluorescence, and a reduction in the release of infectious virus. Even after the drug had been on the persistently infected cells for 72 h, there was still antigen production detectable by immunofluorescence, although the cells no longer hemadsorbed chicken erythrocytes. Ribavirin removal from both types of infection resulted in the renewed synthesis of virus.     

Receptor destruction by viruses of the mumps-NDV-influenza group

A strain each of mumps and Newcastle disease virus and five strains of influenza virus were found to be capable of removing all the receptors for this group of viruses from fowl red cells. Five virus strains were tested for their capacity to inactivate the virus hemagglutinin of human plasma and of egg white. In the case of egg white all strains including mumps and Newcastle disease virus inactivated the inhibitor completely, or nearly so. With plasma the influenza strains inactivated the inhibitor completely but mumps and NDV destroyed only that portion of the complex which effected mumps inhibition. The inhibitor for some strains was destroyed more rapidly than that for others and the sequence in which they were destroyed (inhibitor gradient) was similar, regardless of the strain employed. The inhibitor gradient for egg white was very different from that for plasma and these in turn differed significantly from the receptor gradient for fowl red cells.     

Inhibition of influenza virus multiplication by N-glycosides of benzimidazoles-N

Chloro derivatives of benzimidazole were found to be 2 to 3 times more active than corresponding methyl derivatives in causing inhibition of Lee virus multiplication in chorioallantoic membrane cultures in vitro. The most active benzimidazole derivative thus far tested is 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB); it caused 75 per cent inhibition of Lee virus multiplication in membrane cultures at a concentration of 0.38 x 10(-4)M. On the other hand, 5,6-dimethyl-1-alpha;-D-ribofuranosylbenzimidazole, the moiety present in vitamin B(12), failed to inhibit Lee virus multiplication at a concentration of 35 x 10(-4)M. Other N-glycosides of 5,6-dichlorobenzimidazole were considerably less active than DRB. In single cycle experiments, the degree of inhibition of Lee virus multiplication by DRB in membrane cultures was not dependent on the amount of virus in the inoculum. This compound did not inactivate the infectivity of extracellular Lee virus, had no effect on virus-erythrocyte interaction, did not interfere with the adsorption of the virus by the host tissue, nor affect the release of newly formed virus from the membrane. The inhibitory effect of DRB on Lee virus multiplication, in contrast to that of 2,5-dimethylbenzimidazole, persisted after transfer of infected membranes into fresh culture medium not containing the compound. Both DRB and the 2,5-dimethyl compound caused 99 per cent inhibition of Lee virus multiplication without affecting oxygen uptake of the membrane. Tissue proliferation of membrane pieces in roller tube culture was not significantly affected by DRB at inhibitory concentration, whereas at equivalent concentration the 2,5-dimethyl compound did restrict cellular growth. At higher concentrations, both compounds caused retardation of cell proliferation. This effect was reversible on removal of either compound from the medium. The multiplication of several strains of influenza A and B viruses, i.e. Lee, MB, PR8, and FM1, was inhibited to the same degree by each of the two compounds; DRB was 35 times more active than the 2,5-dimethyl compound relative to each of the strains. DRB caused inhibition of Lee virus multiplication in intact embryonated chicken eggs and in mice without causing significant signs of toxicity in either host. Some of the implications of these findings are discussed in relation to the mechanism of the inhibition of influenza virus multiplication.     

STUDIES OF THE HEMOLYSIS OF RED BLOOD CELLS BY MUMPS VIRUS : II. THE RELATIONSHIPS OF HEMAGGLUTINATION, VIRUS ELUTION, AND HEMOLYSIS

The relationship between hemagglutination and hemolysis by the mumps virus has been studied under conditions which affect (a) the receptors of chicken red cells and (b) the adsorption and subsequent elution of the virus from these cells. The results show that the hemolytic action of the virus appears to involve some of the same receptor areas of erythrocytes that are implicated in hemagglutination. Materials such as allantoic fluid, egg white, and red cell extract, which inhibit the agglutination of chicken red cells by mumps virus, also interfere with its hemolytic activity. Of these inhibitors, egg white and red cell extract, which are readily destroyed by the virus during incubation at 37°C., exert a greater antagonistic effect on hemagglutination than on hemolysis. Heated mumps virus or unheated influenza virus interferes with the hemolysis of red cells by untreated mumps virus. Though hemolysis takes place during elution of the virus after its adsorption on the red cell, the processes are apparently distinct. The hemolytic activity is easily affected by certain conditions of pH and temperature which have no effect on the ability of mumps virus to adsorb on and elute from red cells.     

A CARRIER STATE OF MUMPS VIRUS IN HUMAN CONJUNCTIVA CELLS : I. GENERAL CHARACTERISTICS

Mumps virus produced a carrier state in human conjunctiva cells that was maintained for more than 100 subcultures over a period of 3 years. Antiserum in the medium was not required. The virus had little apparent effect on the cells which grew at a rate similar to uninfected control cells. Mumps virus was regularly found in the culture medium at levels about 0.9 log higher than the cell-associated virus. When first tested after 30 subcultures, the virus was found to have lost its cytopathogenicity for cells ordinarily susceptible to mumps virus, but was identifiable as mumps virus by neutralization with specific antiserum. Use of fluorescein-labeled antiserum revealed that 80 to 95 per cent of cells in the carrier cultures contained mumps virus antigen. The antigen was concentrated in a few sharply circumscribed, discrete masses in the cell cytoplasm rather than in many granules throughout the cytoplasm as is characteristic of cell infection by cytopathogenic mumps virus. The carrier cultures were resistant to the destructive effect of a cytopathogenic line of mumps virus, but showed little resistance to the cytopathogenic effect of vesicular stomatitis, Sendai, or Newcastle disease viruses.     

Inhibition of influenza virus multiplication by alkyl derivatives of benzimidazole. II. Measurement of inhibitory activity by hemagglutination titrations

The activity of compounds which inhibit the multiplication of influenza virus can be measured in chorioallantoic membrane cultures in vitro by means of hemagglutination titrations on the medium. Studies on the reproducibility of virus reproduction in membrane cultures have revealed the major variables which affect the results and thus have led to the development of a precise technique. Under strictly controlled experimental conditions, the extent of reproduction of the virus in membrane cultures is predictable within narrow limits of variation. With 10(5.5) EID(50) of influenza B virus, Lee strain, and 5.75 cm.(2) of chorioallantoic membrane per ml., the ratio of infective virus particles to susceptible allantoic cells appears to be approximately 1:28. Under these conditions, the evidence indicates that two cycles of multiplication occur and nearly maximal hemagglutination titers are found with culture medium at 36 hours. The extent of the deviation in the absolute titer in different experiments was only 0.112 log unit. At a concentration of 0.0017 M, 2,5-dimethylbenzimidazole caused inhibition of the multiplication of influenza B virus, Lee strain, which persisted for at least 70 hours as measured by hemagglutination titrations on the culture medium. The degree of inhibition was closely comparable to that demonstrated by infectivity titrations on the membrane at the end of the first cycle of virus reproduction (1).     

AN INVESTIGATION OF THE ETIOLOGY OF MUMPS

1. From four out of six specimens of saliva from six cases of mumps in the early stages of the disease, a filterable cytotropic virus has been obtained which induces in M. rhesus monkeys, following inoculation of the parotid glands through Stensen''s duct, an acute, non-suppurative parotitis analogous to mumps. 2. This virus has not been found in normal saliva, nor does it correspond to any known virus with which we are familiar. 3. The virus is free of demonstrable microorganisms including spirochetes. 4. It is judged that this virus is the causative agent of mumps.     

CHEMICAL STUDIES ON HOST-VIRUS INTERACTIONS : III. TRYPTOPHANE REQUIREMENTS IN THE STAGES OF VIRUS MULTIPLICATION IN THE ESCHERICHIA COLI-T2 BACTERIOPHAGE SYSTEM

The inhibition of virus multiplication by 5-methyl tryptophane can be specifically reversed by tryptophane. The conditions of reversal indicate that 5MT specifically interferes with tryptophane utilization. The tryptophane requirements of virus multiplication appear to exist throughout the latent period and determine the time of lysis and amount of virus liberated. A latent period may be interrupted for 15 minutes or more and be resumed on addition of tryptophane. Extended inhibition with 5MT results in a somewhat variable "killing" effect, the extent of which determines aspects of the reversal of the inhibition by tryptophane. The implications of these phenomena have been discussed.     

Studies on the hemolysis of red blood cells by mumps virus. IV. Quantitative study of changes in red blood cell lipides and of virus lipides

Hemolysis of chicken red blood cells by mumps virus is associated with the release of sphingomyelin from the stromal lipoprotein and the destruction of 65 per cent of the sphingomyelin of the red cell stroma. However, the virus had no effect on isolated phosphatides extracted from the erythrocytes. The hemolytic action of the virus and changes in sphingomyelin content of the erythrocytes fail to occur at a pH of 6.0. The viral hemolysis of human erythrocytes is not associated with similar alterations in their content of sphingomyelin. The absence of lecithin from sheep erythrocytes, which are also lysed by mumps virus, is additional evidence that a viral lecithinase is not associated with the hemolytic property of mumps virus. Mumps virus concentrated from the amniotic fluid of viral infected chick embryos contains about 7 per cent phosphatide, 60 per cent of which is sphingomyelin.     

Interferon-gamma production closely related to antibody production in lymphocyte cultures stimulated with mumps virus

Enzyme-linked immunosorbent assay (ELISA) antibody to mumps virus and virus specific interferon (IFN)-gamma production were investigated in lymphocyte cultures stimulated with mumps virus before and after immunization with live mumps vaccine. Synthesis of immunoglobulin (Ig) M but not Ig G was enhanced after vaccination. Spontaneous production of mumps ELISA antibodies in lymphocyte culture increased after vaccination and substantially higher levels of antibodies were produced when lymphocytes were stimulated with mumps virus after vaccination. The production of mumps ELISA antibodies was closely related to IFN-gamma production (r = 0.326, p less than 0.01) but not to IFN-alpha production (r = 0.084, p greater than 0.05).     

Studies of delayed hypersensitivity in vitro. II. Delayed hypersensitivity in experimental mumps virus infections

Guinea pigs experimentally infected with mumps virus develop a delayed, hypersensitive skin reaction following the intradermal injection of heat-inactivated mumps virus. This in vivo hypersensitivity is accompanied by a state of cellular hypersensitivity which can be demonstrated in vitro by the addition of mumps viral antigen to cultures of splenic macrophages, following which they become less motile and undergo lysis. These observations support the hypothesis that the state of hypersensitivity which develops early in mumps virus infections may have a role in the pathogenesis of the disease.     

IMMUNITY IN MUMPS : II. THE DEVELOPMENT OF COMPLEMENT-FIXING ANTIBODY AND DERMAL HYPERSENSITIVITY IN HUMAN BEINGS FOLLOWING MUMPS

1. A specific antibody, demonstrable by the technique of complement fixation, regularly appears, or increases in concentration, in the sera of human beings during an attack of mumps or during convalescence. 2. Specific dermal hypersensitivity, demonstrable by the injection of heat-inactivated mumps virus, has been shown to develop in 6 human beings after recovery from mumps. 3. Complement-fixing antibody and the hypersensitive state also emerge as a result of clinically inapparent infection with the virus of mumps. 4. These two phenomena are apparently unrelated in respect to immunologic mechanisms. 5. The data presented indicate that the complement fixation test should prove of value both in diagnosis and in the determination of immunity. 6. The skin test for dermal hypersensitivity, on the other hand, becomes positive after recovery and therefore would appear to be useful only as an index of resistance.     

Studies of hemolysis of red blood cells by mumps virus. III. Alterations in lipoproteins of the red blood cell wall

Evidence from microscopic studies indicates that hemolysis caused by the mumps virus hemolysin is a chemical type of hemolysis. Chromatographic analyses of the reaction mixture of erythrocytes and mumps virus following hemolysis indicate that hemolysis is not due to the action of a lecithinase A and that lysolecithin does not play a part in this process. The alteration of a component of the erythrocyte similar to sphingomyelin suggests that some of the phosphatides other than lecithin may be either directly or indirectly affected in the process of hemolysis of red blood cells by the mumps virus.     

Studies on the psittacosislymphogranuloma group. III. The effect of aureomycin on the propagation of virus in the chick embryo

The findings presented indicate that aureomycin could become associated with tissue of the chick embryo by both hematogenous distribution and direct adsorption. Treatment of chick embryos infected with MP virus with 1 mg. of aureomycin by the allantoic route caused an inhibition of virus growth in the allantoic membrane. The drug had no effect on "inert" virus, and appeared to have little effect on adsorption of virus to host tissue. Complete inhibition of growth during the time interval corresponding to the first cycle of multiplication could be achieved only if the drug was administered within 6 to 8 hours after virus inoculation. Partial inhibition of virus multiplication could be achieved even if the administration was delayed as late as 24 hours after infection. In these experiments the chief role of the antibiotic appeared to be one of virustasis reflected in a prolongation of the latent period (non-infectious phase). The virus was able to resume its growth when a critical low level of the drug in the allantoic membrane was reached. When infectivity titrations were carried out using various tissues and organs of treated and untreated embryos, it was found that no virus was detectable in the brains of treated embryos as late as 192 hours after inoculation of virus. This was in contrast with the findings in allantoic membranes and livers of such embryos; these organs showed virus at 120 and 144 hours, respectively. In untreated controls, virus appeared in membranes at 24 hours, in the liver at 48 hours, and in the brain at 72 hours.     

Studies of influenza virus infection in the chick embryo using fluorescent antibody

As evidenced by specific staining with fluorescent antibody, the major sites of multiplication of the PR8 and Lee B strains of influenza virus in chick embryos injected by the amniotic route were in the cells lining the amnion and in the epidermal and pharyngeal epithelium. Varying amounts of virus were also present in the epithelium of the allantois and less frequently in the peritoneum. No virus was detectable in any of the other tissues of 25 embryos injected between the 7th and 11th days of incubation and examined 48 hours later. Three out of five of the embryos inoculated with the PR8 strain of influenza virus on the 12th day of incubation, on the other hand, showed in addition extensive involvement of the cells lining the respiratory tract. Specific staining of the tissues was first detectable when the ID₅₀ of the amniotic fluids attained a level of greater than 4.5, which corresponded to the time of the appearance of hemagglutinins. With the inocula used this was generally achieved sometime between the 18th and 24th hour of the infection with the PR8 strain of virus and between the 24th and 48th hour of the infection with the Lee B strain of virus. Cytologically, the multiplication of the influenza viruses was characterized by a diffuse type of immunospecific staining which was first detectable in the nuclei and later in the cytoplasm of the cells. The infection progressed rapidly and despite the restricted distribution of the viruses resulted in the death of the embryo in from 3 to 6 days. The results obtained in the present experiments are compared with the findings previously reported in similar studies of mumps virus (6).     

ON THE ROLE OF RIBONUCLEIC ACID IN ANIMAL VIRUS SYNTHESIS : II. STUDIES WITH RIBONUCLEASE

Ribonuclease is a highly active inhibitor of vaccinia virus multiplication in vitro in the chorioallantoic membrane removed from embryonated chicken eggs. It is also a highly active inhibitor of pock formation by vaccinia and herpes simplex viruses on the chorioallantoic membrane in vivo. Marked inhibitory effects were obtained with 12.5 µg. of RNase. However, complete inhibition was not obtained with several hundred micrograms of the enzyme. RNase caused no inactivation of the infectivity of vaccinia virus particles but it had a marked inhibitory effect on multiplication of this virus when administered many hours after infection of host cells had occurred. RNase also failed to inactivate the infectivity of herpes simplex virus particles. The results obtained indicate that ribonucleic acid is necessary for the multiplication of two DNA-containing viruses; i.e., vaccinia and herpes simplex.