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The shut-off of RNA synthesis in chicken embryo cells, after infection with vesicular stomatitis virus, is partially due to a reduced capacity of the infected cells to transport uridine. Permeability to uridine decreases exponentially after infection. This loss of ability to transport uridine may be caused either by structural components of the input virions or may result from the expression of the viral gene products. In the latter case, only minor levels of viral transcription is sufficient to modify cellular permeability, since, even at low multiplicities, RNA minus temperature-sensitive (ts) mutants of vesicular stomatitis virus bring about a significant diminution of uridine incorporation in cells infected under nonpermissive conditions. Experiments with mutants of group III suggest that the M protein of the viral envelope may play a role in the sequence of events that modifies uridine transport. In addition to this cause of the diminution of incorporation of uridine by infected cells, another mechanism is noted which requires protein synthesis.

The sensitivities of selected neurotropic arboviruses (St. Louis encephalitis, Western equine encephaltis, and La Crosse viruses) to human interferon and polyriboinosinic-polyribocytidylic acid (poly I-poly C) were determined in primary human fetal glial cell cultures. The sensitivities were measured by an end point (inhibition of virus yield). All of the viruses were sensitive to human interferon and poly I-poly C; St. Louis encephalitis and Western equine encephalitis viruses were inhibited at concentrations of interferon actually measured in the brains of persons who died from these infections. For St. Louis encephalitis and Western equine encephalitis viruses, viral replication and induction of interferon varied among cell cultures derived from individual fetuses. The present experiments may provide an approach toward understanding the problem of different host susceptibilities to neurotropic arbovirus infections.

An abortive infection of a rabbit cornea cell line (RC-60) by vesicular stomatitis virus (VSV), yielding less than 1 PFU/cell, was converted to a productive infection, yielding 1,900 PFU/cell, when cells were superinfected with vaccinia. Studies on the synthesis of VSV-directed RNA in RC-60 cells suggest that the abortive infection by VSV alone may be due in part to (i) a limited production of 40S virion RNA and (ii) a markedly reduced activity of virion-bound transcriptase activity in RC-60 cells compared to the activity in mouse L cells, a permissive host for VSV. No recognizable VSV structures, except a small amount of viral core structures, were produced by the abortive infection. In contrast, double infection of RC-60 cells with VSV and vaccinia in the presence of hydroxyurea resulted in the production of infective B particles of VSV. Although the function supplied by vaccinia responsible for the productive replication of VSV in double infected RC-60 cells has not been identified, metabolic inhibitor studies indicate that continuous vaccinia-dependent RNA synthesis is required for maximal production of infective VSV. The possibility is considered that vaccinia may supply a product or function required for VSV replication which is ordinarily supplied by the host but which is lacking in RC-60 cells.

Interferon induces protection of enucleated BSC-1 cells against infectious vesicular stomatitis virus production if cells are treated before, but not after, enucleation.

Disulfiram at concentrations between 0.1 and 0.3 mM inhibits the multiplication of Semliki Forest virus (SFV), fowl plague virus (FPV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and pseudorabies virus (PRV), when administered 1 hour before and during adsorption. There is, however, no inhibition of virus multiplication, when the drug is added after adsorption onto chick embryo cells. Disulfiram interferes neither with the receptors of the virus nor of erythrocytes, and it does not prevent virus adsorption. Possibly an early step in virus multiplication is affected by disculfiram. Infected cells once treated with the drug recover after some time of incubation in an ingibitor-free medium. The inhibitory state can be maintained, however, if relatively low doses of disulfiram are present in the culture medium also after adsorption. Disulfiram has no effect on macromolecular synthesis of the host cells. It has, however, a marked affect on membrane function. While virus multiplication is readily inhibited by disulfiram when chick embryo or BHK cells were investigated, virus multiplication in HeLa cells is almost resestant against the action of disulfiram.

Electron microscopy observations of purified Bryan high-titer Rous sarcoma virus (BH RSV) using the freeze-drying technique showed that progeny made in the absence of a helper virus lacked visible surface projections or spikes. Phenotypic mixing experiments employing BH RSV and a thermolabile mutant of vesicular stomatitis virus, tl 17, yielded no evidence of pseudotype formation. Since tl 17 is known to be defective for an envelope glycoprotein, the lack of successful phenotypic mixing with BH RSV is consistent with the observed absence of viral spikes.

Infectious B virions of vesicular stomatitis virus were 100% lethal to BHK(21) (baby hamster kidney) cells when infecting alone, and persistent noncytocidal infection could not be achieved with cloned B virions alone. However, a mixture of B virions and homologous, short, defective, interfering particles (T particles) of a temperature-sensitive mutant of the virus regularly established persistently infected, noncytocidal carrier cultures. A long T particle was generated during establishment of the carrier culture; we show that this long T particle can establish and maintain persistent noncytocidal infection even when it infects cells along with virulent wild-type B virions. This long T particle causes the production of wild-type B virions with greatly reduced virion transcriptase (EC 2.7.7.6; RNA nucleotidyltransferase) levels when coinfecting the same cells, so it appears to prevent cytopathology by regulating virus transcription. The implications of these findings for rabies and other slowly progressing noncytocidal infections are discussed.

The requirement of the presence of a nucleus for the replication of vesicular stomatitis virus and influenza virus has been examined by following the growth and development of these viruses in enucleate BS-C-1 cells. Vesicular stomatitis virus replicates normally in enucleate cells with the rate of production of infectious virus, the amount of virus-specific protein synthesis, and the type of proteins produced being essentially the same in nucleate and enucleate cells. Influenza virus does not replicate in enucleate cells, no virus gene products can be detected, and there is no inhibition of cellular protein synthesis.

Electron microscopy showed that commerical poly I: C consisted of molecules varying in length from less than 0.05 nm to more than 5 nm and also in morphology . To clarify the relationship between its molecular size and interferon-inducing activity, poly I: C was sonicated or fractionated by sucrose density gradient centrifugation, and the molecular length distribution and interferon-inducing activity of each preparation was determined in vivo and in vitro. The results showed that (1) poly I : C molecules 0.1-0.3 nm long were the most effective for interferon induction; (2) sonication of poly I : C reduced its molecular length and also the interferon-inducing activity, the degree of reduction varying in different fractions; and (3) the interferon-inducing activity of poly I: C of 0.1-0.3 nm obtained by sucrose density gradient centrifugation was higher than that poly I: C of corresponding length prepared by sonication.

After an initial acute infection with cell killing, chicken or duck embryo fibroblasts infected in culture with reticuloendotheliosis viruses set up a chronic infection with no cell killing or morphological transformation. Essentially all of the chronically infected cells produced virus. The virus production was not sensitive to cytosine arabinoside or mitomycin C as was virus production in an acute infection. The chronically infected cells had a strong group-specific resistancto the c.p.e. of superinfecting reticuloendotheliosis viruses. However, they were sensitive to vesicular stomatitis virus and avian leukosis-sarcoma viruses. After double infection, single cells produced reticuloendotheliosis virus and avian leukosis-sarcoma virus.

The effect of low pH's on the development of vesicular stomatitis virus (VSV) was studied. L cells infected with VSV were incubated at pH 6.6. A 99% inhibition in the yield of infectious particles was observed by comparison with the yield at pH 7.4. Such inhibition was not due to inhibition of viral RNA synthesis, since at pH 6.6 all the known species of VSV RNA molecules were synthesized. Furthermore, all the known species of VSV proteins were also synthesized. However, no viral particles nor nucleocapsids were detected. Raising the pH to 6.9 resulted in the appearance of nucleocapsids and viral particles, although the yield of infectious virus was still inhibited by 90%. The lack of infectivity of these pH 6.9 viral particles was correlated with their inability to promote primary transcription. The hypothesis that low pH's alter the correct positioning of the viral proteins into the cell membrane is presented.

L2 cells infected with vesicular stomatitis virus under single-cycle conditions have been studied by scanning electron microscopy after preparation by the critical point drying technique. Three dimensional images of intact cells show bullet-shaped vesicular stomatitis virus virions budding singly and in radiating clusters both from the plasma membrane between cellular microvilli and from the sides of microvilli. Virus-induced cytopathic effects observed by scanning electron microscopy include intermeshing of microvilli, loss of filipodia which attach cells to the substrate, and rounding up and detachment of infected cells from the substrate.

Infection of enucleated TC-7 monkey cells with rabies virus resulted in the synthesis of virus-directed RNA and the production of rabies antigens but not of infectious virus. The yield of infectious vesicular stomatitis virus from enucleated TC-7 cells, on the other hand, was almost as high as that from intact cells. Inhibition of the mitochondrial functions of enucleated cells by treatment with ethidium bromide did not influence the development of rabies antigens or the production of infectious vesicular stomatitis virus.

Cells infected with temperature-sensitive (ts) mutants of complementation group V of vesicular stomatitis virus (VSV) give an enhanced yield at nonpermissive temperature when co-infected or superinfected with UV-irradiated virus. Virions produced in these mixed infections are temperature sensitive and do not complement ts V45. Rescue of group V mutants is multiplicity dependent. It can occur in the presence of cycloheximide; kinetics of rescue are similar in the absence or in the presence of the drug. Rescue is due to nongenetic complementation and is interpreted as a trigger effect on maturation of a small quantity of biologically active protein V molecules provided by UV-irradiated virus. These results are comfirmed by rescue of ts V45 by UV-irradiated, defective, interferring T particles.

Cytopathological effects by vesicular stomatitis virus (VSV) infection were studied in several cell lines. Marked polykaryocyte formation was observed in monolayers of certain strains of BHK-21 cells infected with VSV. The BHK-21-KB cells were found to be the most susceptible to virus-induced cell fusion. This type of cell fusion was related to intracellular growth of the viruses, and strong cytolytic changes were found to occur following the development of large multinucleated giant cells. The cell-fusing activity was associated with the infectivity of VSV and was neutralized by anti-VSV immune serum. The viruses irradiated for 20 minutes or heated at 60 degrees C for 10 minutes lost completely both infectivity and cell-fusing activity. These experimental results indicate that virus replication was responsible for fusion of BHK cells.

Two human lymphoid cell lines, Raji and Namalwa, and seven somatic cell hybrids between these two cells were tested for spontaneous interferon production. Namalwa and three of the hybrids produce interferon while the four other hybrids, like Raji, do not. Though the Namalwa line is relatively resistant to superinfection with the Epstein-Barr virus (EBV), all hybrids are as susceptible to EBV as the Raji parent. High extracellular concentrations of interferon likewise had little effect on EBV superinfection of Raji or the hybrid cells and it is possible that Raji produces some factor that antagonizes the action of interferon to allow virus expression.

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.

The enhancement of vesicular stomatitis virus plaques on human embryonic lung cells in the presence of Tween 80 or Aquasol A was studied to determine the optimal conditions for the enhancement. Enhanced numbers and sizes of vesicular stomatitis virus plaques occurred when Aquasol A or Tween 80 was added to the cell culture 30 min before virus adsorption but not when added after adsorption. These substances did not have a direct effect on the virus and did not have an effect on virus adsorption or penetration. A few other viruses and cell systems were also studied to determine if enhancement would extend to other viruses and cell systems. The cell system seemed to be important since enhancement of vesicular stomatitis virus plating efficiency did not occur on chicken embryo cells. However, the virus was also important since vaccinia virus plating efficiency was not enhanced on the human embryonic lung cell. The greatest enhancement encountered was the increase in the plating efficiency of Friend leukemia virus on S+L- cells.

Cytopathic changes and virus-specific antigens developed in, then disappeared from, mouse fibroblasts infected by a strain of human cytomegalovirus (CMV), but their disappearance was delayed in cells treated with idoxuridine prior to infection. The replication of vesicular stomatitis virus and herpes simplex virus was restricted in human CMV-infected mouse cells as long as human CMV-specific antigens were present. Virus-specific antigens could be induced by treatment with idoxuridine or arginine deficiency in mouse cells which had previously turned "negative".

The morphogenesis of vesicular stomatitis virus (VSV) in three reptilian cell lines (turtle heart, gecko lung, and viper spleen) was studied by thin section electron microscopy. Simultaneous growth studies were conducted in reptilian, chick embryo fibroblast, and BHK/21 cells to compare the yields of infectious VSV during serial passages. The mean length of gecko lung cell VSV measured from electron micrographs is statistically significantly shorter than that of VSV replicating in other reptilian systems studied. This observation, along with comparative growth studies, suggests the predilection of gecko lung cells to form "auto-interfering" truncated "T" rather than infectious "B" VS virions.

Pseudotypes of vesicular stomatitis virus were prepared with avian sarcoma viruses and avian leukemia viruses representing five different subgroups. These pseudotypes display a host range restricted to that of the avian tumor virus when assayed on avian cells and are neutralized by subgroup-specific antisera. The efficiency of penetration of mammalian cells was assayed by using these vesicular stomatitis virus pseudotypes. Pseudotypes of avian tumor viruses belonging to subgroup D and of B77 virus were able to plate on mammalian cells with a high efficiency, whereas pseudotypes of other strains were not. The efficiency of penetration of the vesicular stomatitis virus pseudotypes was 10-2-to 10-3-fold higher than the efficiency of transformation of the corresponding avian tumor virus strain assayed on mammalian cells, suggesting that there are postpenetration blocks to the expression of transformation in these cells.

Rifampin reversibly inhibits the intracellular replication of a VSV mutant, whereas a revertant variant selected from this viral population, as well as the wild strain, are not affected by this drug. Rifampin inhibits transcriptase activity of the sensitive mutant only and, consequently, total viral RNA synthesis decreases significantly in the cells.

The frequency of naturally occurring transplacental infection of swine with porcine parvovirus (PPV) and one of the possible consequences of such infection--the presence of PPV in cell cultures prepared from fetal tissues--were investigated. Transplacental infection was indicated by the presence of high titers of hemagglutination inhibiting (HI) antibody for PPV in serums of 0-day-old, hysterectomy-derived, colostrum-deprived pigs of 3 of 82 litters. All letters were farm-raised dams. Moreover, cell cultures prepared from 3 of 49 lots of fetal porcine kidneys (FPK) collected from an abattoir during an interval of 14 months were found contaminated with PPV. Because each lot was usually comprised of kidneys from 2 litters, the latter finding suggests that 3 of approximately 98 litters were infected. Prior infection of FPK cell cultures with PPV resulted in only slight interference of replication of other selected viruses; i.e., porcine enterovirus (PEV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), and hemagglutinating encephalomyelitis virus (HEV). Moreover, PPV and HEV were propagated in the same cell cultures during 5 serial passages of the viruses. In contrast, when copropagation of PPV and VSV was attempted, PPV was not detected after the 2nd serial passage.

Initiation of treatment with potent interferon preparations (6-4 x 10-6 units per dose) 4 days after intranasal inoculation of VSV (at a time when virus has already multiplied in the brains of most mice) resulted in a marked increase in mouse survival. In a series of 6 experiments only 11 to 18% of control mice survived whereas 52% of interferon treated mice survival. These results suggest the usefulness of exogenous interferon even when treatment is begun late in the course of an acute virus disease.

The virulence of temperature-sensitive mutants of vesicular stomatitis virus (VSV) injected subcutaneously into newborn hamsters was positively correlated with their tendency to generate revertants and with their leakiness in cultured hamster embryo fibroblasts maintained at 37 degrees C, the measured body temperature of the animals under our experimental conditions. The complementation group of the mutants seemed important only in that it tended to determine reversion frequency and leakiness. One non-reverting group I mutant (T1026), however, was much less virulent than would be expected from its extreme leakiness at body temperature. The disease produced by the less virulent mutants was characterized by neurological symptoms and led to delayed death, unlike the rapid deatth produced by virulent mutants. Infectious virus could be found in higher titres in the brains than in peripheral organs of such animals (with ratios as high as 10(8)). This neurotropism was not correlated with the complementation group of the mutant but was shown to be the consequence of survival for more than 3 days after injection. Age was not responsible for the effect. Animals injected at birth with T1026 were completely resistant to subcutaneous superinfection with the highly virulent wildtype virus HR at 3 to 4 days, though non-T1026-protected animals were completely sensitive. When HR was injected intracerebrally at 3 to 4 days, the T1026-protected animals allowed replication to high titres in the brain but not in peripheral organs, whereas non-T1026-protected animals allowed replication to high titres in both brain and in peripheral organs. We suggest from these results that the observed neurotropism is produced by a resistance mechanism operative in peripheral organs but not in the brain; this resistance develops rapidly in newborn animals on exposure to virus and clears virus from the peripheral organs leaving it in the brain. It is possible that our effect represents a controlled and accelerated induction of the classical peripheral resistance of animals to various viruses which normally develops with age.

Vesicular stomatitis virus propagated in and released from Aedes albopictus cells had the normal complement of viral proteins; the glycoprotein contained carbohydrate but no sialic acid. These virions had markedly reduced hemagglutinating activity and exhibited a very high ratio of physical particles to infectious virus. In vitro sialylation of vesicular stomatitis virions grown in mosquito cells resulted in a 100-fold increase in both infectivity and hemagglutination titers to levels approaching those of virus grown in BHK-21 cells. These experiments provide an example of host-controlled modification of viral infectivity.

The oligosaccharides of the membrane glycoproteins of Sindbis virus, vesicular stomatitis virus, and Rous sarcoma virus were compared on the basis of apparent size and sugar composition. It appears that each virus acquires a different set of oligosaccharides during growth in a single type of cell.

A local antiviral effect can be observed when (poly rI)-(poly rC), bound to Visking discs by u.v. irradiation, is incubated with monolayers of human foreskin fibroblast cells. Radioactive labelling of cytosine residues in (poly rI)-(poly rC) with -125I, has provided a much more sensitive method for determining the fate of the insoluble (poly rI)-(poly rC) than has been available hitherto. The antiviral effect is not related to the amount of (poly rI)-(POLY RC) present on the insoluble support but rather to the amount of polynucleotide lost from the support during incubation. Treatment of (poly rI)-(poly rC) which had been bound to cyanogen bromide-activated Sepharose with eigher dilute alkali or pancreatic ribonuclease released virtually all the polynucleotide. A small amount of (poly rI)-(poly rC) is released from the insoluble matrix in the presence of serum-free Minimum Eagle's Medium.

An interaction between sarcoma-180/TG cells and vesicular stomatitis virus in adult mice resulted in the rapid onset of extensive mortality. This interaction, termed lethal synergy, occurred only at early stages of ascites induction in animals with no prior virus contact. A significant sparing effect conferred by the serotonin antagonist dibenamine was reversed by the administration of serotonin. The cause of death was not determined, but a mechanism involving hypersensitivity is indicated.