Suppression of Growth of Herpes Simplex Virus in Chick Embryo Fibroblasts at 40 C

Four strains of herpes simplex virus tested all showed inability to form plaques in chick embryo fibroblasts (CEF) at 40 C, while no such suppression of growth was observed with WEE, vaccinia or JE virus in the same cells. The suppression was not due to a complete inhibition of viral growth, because virus-infected CEF bottle cultures consistently showed a small but definite increase of virus titer in 24 hr. When CEF monolayers adsorbing herpes virus were placed at 40 C, the number of infective centers decreased gradually; however, this decrease was much slower than the degradation of free virus at this temperature. Transfer of virus-infected CEF bottle cultures from 35 C to 40 C at any time during a one step growth cycle promptly slowed down subsequent virus replication. When virus-infected CEF bottles were incubated first at 40 C for 24 hr and then transferred to 35 C, a new increase in virus titer took place following a short lag. What stage of virus replication is suppressed at 40 C remains yet to be determined.     

Isolation and characterization of heat-resistant (HR) mutants of Newcastle disease virus

Heat-resistant (HR) mutants (MR 70 and HR 74) of Newcastle disease virus (NDV) which exhibited significantly higher thermostability in their infectivity than wild-type virus were isolated and characterized. They differ from each other in their plaque morphology; HR 70 produces small turbid plaques, whereas those of HR 74 are large and clear. Cytopathogenicity of these mutants is much lower than that of the wild-type virus in cultured cells such as CEF, LLCMK2 and HeLa cells. Moreover, these HR mutants exhibited extended mean embryo survival times. Synthesis of cellular RNA's and proteins in cells infected with HR mutants was not significantly reduced under conditions in which synthesis of these macromolecules was strongly reduced in cells infected with wild-type virus. No significant differences were observed between HR mutants and wild-type virus in their other phenotypic characteristics such as the capacity for interferon production, growth characteristics at a low multiplicity of infection, and cleavage of viral glycoproteins in infected cells. From these findings, it was suggested that the inhibitory effect of virus infection on cellular macromolecular synthesis is a possible determinant of cytopathogenicity of NDV.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

Plaque Formation by Mumps Virus and Inhibition by Antiserum

Boston and ABC strains of mumps virus produced plaques approximately 1.0 mm in diameter in monolayers of BGM cells. The plaques were circular and either clear or target-like in form. Ricki strain virus produced plaques of similar size and form but, in addition, a red plaque was observed with this agent. The vaccine strain of mumps virus, Jeryl Lynn, produced minute clear plaques approximately 0.3 mm in diameter. Incorporation of diethylaminoethyl (DEAE)-dextran into the overlay medium did not affect the size difference between Jeryl Lynn plaques and those of the other strains. However plaques of the Jeryl Lynn and Ricki strains were more easily visualized when the overlay medium contained 400 mug/ml of DEAE-dextran. Simultaneous titration by plaque formation and roller tube infectivity showed that these two methods were of equal sensitivity. Virus neutralization by antibody was demonstrated by plaque reduction. Rise in antibody titer was observed in sera from human and animal infection, human vaccination, and rabbit immunization.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Plaquing of Toxoplasma gondii in Secondary Cultures of Chick Embryo Fibroblasts

A tissue culture system that employed chick embryo fibroblasts was described for plaquing of the obligate intracellular parasite Toxoplasma gondii. High plaquing efficiency and reproducibility were accomplished by the use of secondary rather than primary cultures and the use of toxoplasma obtained from disrupted peritoneal cells of mice infected 48 hr earlier. The monolayers were cultured in a special medium which maintained the fibroblasts until the maximal number of plaques was produced. Optimal plaque formation was obtained in 5 days, and the plaques were easily counted macroscopically.     

Ultraviolet enhanced reactivation of a human virus: effect of delayed infection

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.     

Fungal nucleic acids as interferon inducers.

Nucleic acids isolated from the fungi Aspergillus niger x11, Piptoporus betulinus and Ganoderma applanatum reduced the number of vaccinia virus plaques in chick embryo fibroblast (CEF) tissue culture and when administered intravenously to white mice protected them against lethal infection with tick borne encephalitis virus strain K5 (TBE). In CEF tissue culture the nucleic acids of the studied fungi were found to induce small but detectable amounts of a substance with the character of interferon. In vivo only ribonucleic acid from G. applanatum induced a substance showing interferon properties in the spleen of mice.     

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.     

Plaque Formation by 55 Rhinovirus Serotypes

Plaque production by 55 rhinoviruses in several lines of HeLa cells is reported. Forty-nine types produced macroscopic plaques under the conditions described previously employing 30 mM MgCl(2) and 30 mug/ml of DEAE-dextran in overlay and M HeLa cells. Basically three kinds of plaques were observed: clear large and intermediate sized plaques and small turbid plaques. Five rhinoviruses produced plaques only in a sensitive clonal line isolated from the M HeLa line. Rhinovirus type 4 produced plaques after the pretreatment of cells with actinomycin D. Enhancement of plaque formation by MgCl(2) has been demonstrated to date with 17 rhinoviruses. Some rhinoviruses were enhanced either by DEAE-dextran or by dextran sulfate in the overlay. No inhibitors were found in any of 10 bovine sera tested. Rhinovirus type 2 was visualized inside HeLa cells by electron microscopy and there appeared to be a very high ratio of physical particles per infectious unit of virus.     

Endogenous ecotropic mouse type C viruses deficient in replication and production of XC plaques.

Endogenous ecotropic type C viruses were induced by iodedeoxyuridine from nontransformed and chemically or spontaneously transformed clones of the C3H/10T1/2 cell line. Viruses produced by cells of certain transformed clones were N-tropic and formed large XC plaques. In contrast, viruses produced by nontransformed C3H/10T1/2 cells were not detectable in the XC plaque test. These XC- viruses infected mouse cells with high efficiency, as shown by the induction of murine leukemia virus group-specific antigens in infected cells, but virus production, as determined by DNA polymerase-containing particles, was extremely low. Upon growth in certain mouse cells these replication-deficient, XC(-) viruses converted to type C viruses that were similar in XC assays to N-tropic AKR virus (XC+).     

Persistence of mumps virus in mouse L929 cells

The characteristics of a persistent infection of L929 cells with mumps virus (MuV) is presented. The persistent infection (L-MuV cells) was regulated by interferon (IFN) produced endogenously and almost all the properties showed that the carrier culture was maintained by horizontal transmission of the virus. Small-plaque mutants, but not temperature-sensitive variants, were selected during the persistent infection. MuV released from L-MuV cells (MuV-pi) replicated efficiently in L929 cells, while infection of L929 cells with the original MuV-o resulted in an abortive infection. The efficient replication of MuV-pi in L929 cells can be explained by the findings that MuV-pi induced IFN more slowly and had lower susceptibility to IFN in L929 cells than MuV-o did. M protein was synthesized to a considerable degree in MuV-pi-infected cells, while it could not be detected in MuV-o-infected cells. By contrast, MuV-pi formed small plaques in Vero cell monolayers and the yield of MuV-pi in Vero cells was lower than that of MuV-o. M protein induced by MuV-pi decayed easily in Vero cells. M protein was considered to be a limiting factor for MuV replication in both cell lines.     

Adaptation of Plaque Assay Methods to the in Vitro Quantitation of the Radiation Leukemia Virus

A modification of the XC cell procedure for murine leukemia virus assay which yields quantitative data over a wide range of virus concentrations is described. By using serial passage of infected cell cultures and reversal of the plating sequence in the XC procedure, titers of radiation leukemia virus (RadLV) were obtained which were about 10-fold higher than those found by using the conventional assay. By using the modified procedure, it was observed that, even at high multiplicities of infection, less than 10% of the cells function as infective centers, although the proportion increases with serial passage. It was also observed that exposure of infected cells to UV light, which is commonly used to make plaques more visible in the conventional XC cell test, inhibits plaque formation in the RadLV system. Substitution of X irradiation for UV exposure improved plaque visibility without loss of sensitivity.     

Plaque Development and Induction of Interferon Synthesis by RMC Poliovirus.

Johnson, Terry C. (University of Minnesota, Minneapolis), and Leroy C. McLaren. Plaque development and induction of interferon synthesis by RMC poliovirus. J. Bacteriol. 90:565-570. 1965.-Plaque development by RMC poliovirus on human amnion cell monolayers was investigated with regard to autointerference and to the effect of acid-agar overlay on plaquing efficiency. The virus was inhibited by acid-agar overlay, thereby exhibiting the d(-) marker typical of attenuated poliovirus strains. In addition, a lack of RMC poliovirus plaque development on HeLa cell monolayers was shown to be the result of an agar inhibitor which could be removed by NaCl extraction. By use of a simplified plaque reduction assay, it was shown that interferon production was responsible for the autointerference phenomenon. Interferon synthesis did not correlate with the ages in vitro of human amnion cell cultures. Fibroblasts originating from the chorionic membrane produced negligible amounts of the inhibitor. Interferon synthesis by human amnion cells infected with RMC poliovirus was inhibited by actinomycin D. The addition of guanidine hydrochloride to infected cultures immediately after RMC poliovirus adsorption markedly inhibited interferon synthesis, although after 2 hr (postadsorption) guanidine had no effect on interferon production.     

Antiviral action of mouse interferon in heterologous cells

Buckler, Charles E. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), and Samuel Baron. Antiviral action of mouse interferon in heterologous cells. J. Bacteriol. 91:231-235. 1966.-The antiviral action of mouse interferon in cell cultures of mouse, hamster, rat, chicken, and monkey origin was investigated. Using a vesicular stomatitis virus (VSV) plaque reduction test, we found that mouse serum interferon, assayed on closely related rat or hamster cells, exerted 5% of its homologous antiviral activity. This activity was characterized as interferon by its temperature of inactivation, trypsin sensitivity, nonsedimentability, stability at pH 2, lack of inactivation by antibody to virus, and inability to be washed off cells. In the more distantly related chicken and monkey cells, mouse interferon had less than 0.1% of its homologous activity. Conflicting reports of heterologous activity of chicken and mouse interferon preparations may result in part from the observed action of noninterferon inhibitors of vaccinia virus. These inhibitors, like interferon, are stable at pH 2. They are present in mouse serum, mouse lung extracts, and allantoic fluid, and they prevent the development of vaccinia plaques when allowed to remain in contact with cells during virus growth. Unlike interferon the inhibitors are removed by adequate washing of cells prior to virus challenge, and they are not active in the VSV assay system. These findings reemphasize the need for thorough characterization of interferon preparations.     

Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus.

Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses. Titers obtained with Mengo virus as challenge were all lower than with VSV. With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold. With Mengo virus-induced interferon the reduction was much greater (about 17-fold). This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus. The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus. This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro.     

Effect of Persistent Fibroma Virus Infection on Susceptibility of Cells to Other Viruses

Shope fibroma virus establishes a persistent cytoplasmic infection in primary (RK) and serially cultivated (DRK(3)) rabbit kidney cells which is accompanied by a morphological alteration of the cells. The response of such cells to superinfection by other viruses was compared with that of control cells by determining plaque production and virus yield of superinfecting viruses. It was found that the growth of other poxviruses, myxoma and vaccinia, was greatly inhibited in the fibroma virus-infected cells, but that of pseudorabies and herpes simplex viruses, which are unrelated deoxyribonucleic acid viruses, was virtually unaffected. The ribonucleic acid (RNA) viruses, poliovirus 1 and coxsackievirus B1, did not produce plaques on either RK or fibroma virus-infected (F-RK) monolayers. However, the growth of several other RNA viruses, vesicular stomatitis virus, encephalomyocarditis virus, Sindbis virus, and Newcastle disease virus, was enhanced in F-RK cells. None of these latter RNA viruses produced any infectious progeny in DRK(3) cells, but they all plaqued on and produced good yields in DRK(3) cells persistently infected with fibroma virus. This phenomenon is termed facilitation. Facilitation results from the infection of DRK(3) cells by fibroma virus. Neither interference nor facilitation were due to changes in the adsorption or eclipse of the superinfecting virus.     

Suppression of herpes simplex virus 1 in MDBK cells via the interferon pathway

Herpes simplex virus (HSV) normally undergoes productive infection in culture, causing cell destruction and plaque formation. Here we characterize an unusual pattern of HSV type 1 (HSV-1) infection in MDBK cells which surprisingly results in suppression of replication, cell recovery, and maintenance of virus. Compared to Vero cells, MDBK cells supported a normal productive infection at a high multiplicity with complete cell destruction. At low multiplicity, HSV also showed an identical initial specific infectivity in the two cell types. Thereafter, the progression of infection was radically different. In contrast to the rapid plaque expansion and eventual destruction in Vero monolayers, in MDBK cells, after initial plaque formation, plaque size actually decreased and, with time, monolayers recovered. Using a green fluorescent protein (GFP)-VP16-expressing virus, we monitored infection in live individual plaques. After early stages of intense GFP-VP16 expression, expression regressed to a thin boundary at the edge of the plaques and was completely suppressed by 10 days. Cells lacking expression then began to grow into the plaque boundaries. Furthermore, following media replacement, individual cells expressing GFP-VP16 could be observed reinitiating infection. The results indicated the production of a potent inhibitory component during infection in MDBK cells, and we show the continued and prolonged presence of interferon in the medium, at times when there was no longer evidence of ongoing productive infection. We exploited the ability of V protein of simian virus 5 to degrade Stat1 and prevent interferon signaling. We established MDBK cells constitutively expressing the V protein with the resultant loss of Stat1. In comparison to the parental cells, infection in these cells now progressed at a rapid rate with expanding plaque formation. We believe the conclusions have significant implications for the study of HSV-1 and interferon signaling both in culture and in animal models.