Chick Embryo Cell Cultures Research Articles

Cells persistently infected with Newcastle disease virus. II. Ribonucleic acid and protein synthesis in cells infected with mutants isolated from persistently infected L cells.

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.

Arbovirus isolations from mosquitoes collected in central Utah in 1967.

Abstract During a 1967 arbovirus survey of Utah's Great Basin. mosquitoes, collected by light traps in Cedar and adjacent Utah Valley, were pooled by species, and assayed by routine procedures in suckling mice and chick-embryo cell cultures. Viruses were identified by neutralization in suckling mice and complement-fixation tests. Culex tarsalis, Culex erythrothorax, Culiseta inornata, and Ades dorsalis constituted 91% of the total 18.878 mosquitoes collected. All 23 arbovirus isolates were from these four species. Eight Hart Park-like isolates were obtained only from C. tarsalis. The remaining 15 isolates were California encephalitis group viruses, seven from C. inornata, six from A. dorsalis. one from C. tarsalis, and one from C. erythrothorax. The best vector of California encephalitis group virus was indicated to be C. inornata by the highest total infection ratio.

Gamma-irradiated Venezuelan equine encephalitis vaccines.

The efficacy of Formalin-inactivated Venezuelan equine encephalitis (VEE) vaccine has been reported to be low for man. Although a live VEE vaccine has been shown to be highly effective for the protection of laboratory workers, local and systemic reactions have occurred in approximately 20% of inoculated individuals. Therefore, studies were initiated in an attempt to produce an inactivated vaccine of high potency with low toxicity. Inactivated VEE vaccines were prepared by exposing virus suspensions to 8 x 10(6) or 10 x 10(6) r of gamma radiation. Irradiated VEE vaccines prepared from virus suspensions produced in Maitland-type chick embryo (MTCE) cell cultures and in monolayer cultures of human diploid strain WI-38 cells were highly immunogenic for mice and guinea pigs. Guinea pigs vaccinated with a series of three inoculations of vaccine (MTCE) survived challenge with at least 10(8.4) mouse intracerebral 50% lethal doses of VEE virus. Irradiated vaccines induced high levels of serum-neutralizing and hemagglutinin-inhibiting antibodies in guinea pigs and rabbits. These findings suggest that ionizing radiation may be effective in the preparation of an inactivated VEE vaccine.

RNA-induced interference with animal virus infection. Dependence on cellular metabolic activity.

Some RNAs, including both single- and double-stranded RNAs, when incubated with chick embryo cell culture induce cellular resistance against viruses. Evidence was now obtained indicating that the induction of cellular resistance by RNA depends on the cellular metabolic activity, especially on the synthesis of cellular RNA and protein. Thus, inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, were found to inhibit the development of an antiviral state when added before, or during the relatively early period of, incubation of the cells with RNA. In the course of induction of cellular resistance, three stages may be distinguished, the priming stage, the developing stage, and the established resistant stage.

Influence of an arbovirus infection (Sindbis virus) on the protein and ribonucleic acid synthesis of cultivated chick embryo cells.

Cultured chick embryo cells infected with a group A arbovirus (Sindbis) showed a decreased synthesis of protein and RNA. Protein synthesis was more markedly depressed than RNA synthesis. Inhibition of protein synthesis started 1 to 1 1/2 hours after infection, and cup to 50 per cent inhibition occurred 6 hours postinfection. Results obtained by using p-fluorophenylalanine, actinomycin D and an interferon preparation suggest that the inhibition of cellular protein synthesis depends on the production of protein(s) coded by the viral genome.

Purification of Semliki Forest virus using disulphide-linked immunosorbents derived from rabbit or fowl antibody.

SUMMARY Semliki Forest virus in the crude supernatant from a culture of chick embryo cells was specifically adsorbed by a disulphide-linked immunosorbent prepared from the γ-globulins of rabbits immunized by Semliki Forest virus grown in mouse brain. Two to 6 ml. of rabbit antiserum of neutralization index 4 to 5 yielded 10 mg. of immunosorbent which bound 1 to 6 × 1010 p.f.u. from 2 to 30 ml. of culture supernatant. Virus, free from most chick cell constituents, could be eluted from the immunosorbents by treatment with bicarbonate/carbonate buffer at pH ii-i. The immunosorbents could be cycled at least five times and about 50 % of the adsorbed virus infectivity recovered at each cycle after the first, from which only 10 to 20 % was recovered. In capacity experiments, immunosorbents derived from chicken antisera against Semliki Forest virus grown in chick cells showed no advantages over those derived from rabbit antisera.

An inactivated eastern equine encephalomyelitis vaccine propagated in chick-embryo cell culture. II. Clinical and serologic responses in man.

An inactivated eastern equine encephalomyelitis vaccine propagated in chick-embryo cell culture. II. Clinical and serologic responses in man.

An inactivated eastern equine encephalomyelitis vaccine propagated in chick-embryo cell culture. I. Production and testing.

Abstract A formalin-inactivated Eastern equine encephalomyelitis vaccine was prepared from the PE-6 strain virus, propagated in primary chick-embryo cell cultures maintained with Medium 199 containing 0.25% human serum albumin. To effect an increase in antigenic mass per unit volume of fluid harvest, we used the pooled fluids from the first tissue-culture passage as maintenance medium for two additional passages. A formalin concentration of 1:2,000 (0.05%) at 37°C was used for inactivation of the virus suspension. Tests on the “in-process” and final vaccine documented the safety and sterility. Tests of potency on the final lyophilized product in hamsters and guinea pigs yielded ED50 values of 0.009 ml and 0.012 ml, respectively. Recent assays have revealed no change in potency after storage of the final product for 17 months at -20°C.

Gamma-Irradiated Venezuelan Equine Encephalitis Vaccines

The efficacy of Formalin-inactivated Venezuelan equine encephalitis (VEE) vaccine has been reported to be low for man. Although a live VEE vaccine has been shown to be highly effective for the protection of laboratory workers, local and systemic reactions have occurred in approximately 20% of inoculated individuals. Therefore, studies were initiated in an attempt to produce an inactivated vaccine of high potency with low toxicity. Inactivated VEE vaccines were prepared by exposing virus suspensions to 8 x 10(6) or 10 x 10(6) r of gamma radiation. Irradiated VEE vaccines prepared from virus suspensions produced in Maitland-type chick embryo (MTCE) cell cultures and in monolayer cultures of human diploid strain WI-38 cells were highly immunogenic for mice and guinea pigs. Guinea pigs vaccinated with a series of three inoculations of vaccine (MTCE) survived challenge with at least 10(8.4) mouse intracerebral 50% lethal doses of VEE virus. Irradiated vaccines induced high levels of serum-neutralizing and hemagglutinin-inhibiting antibodies in guinea pigs and rabbits. These findings suggest that ionizing radiation may be effective in the preparation of an inactivated VEE vaccine.

Effect of proteolytic enzymes on vaccinia virus replication.

Chymotrypsin and trypsin enhance vaccinia virus replication in chick embryo cell cultures. Two phenomena are described: an enhancement of plaque numbers and an increase in plaque size. Low doses of chymotrypsin (1.3μg/4 million cells) acting on the virus particles are responsible for the increase in vaccinia virus plaque numbers. The effect is not due to disaggregation of virus aggregates by the enzyme. Doses of 3 to 10 (μg of chymotrypsin per culture have an effect on chick embryo cell monolayers. A linear dose response is found when plaque size is measured as a function of the dose of chymotrypsin. Preliminary studies indicate that some of the enzyme becomes cell-associated. When one replication cycle was followed the final yield of plaque forming virus from both enzyme treated and control cells was the same. However, the process of replication in enzyme treated monolayers was accelerated by about 5 hours. The enzyme appears to have an effect on some early event in virus replication.

Accelerated plaque formation by fowlpox virus in the presence of chymotrypsin.

Chymotrypsin enhanced fowlpox virus plaque formation in chick embryo cell cultures. A simplified plaque assay for fowlpox virus is described. Plaques were produced in 3 days when chymotrypsin was included in a serum-free fluid overlay. Plaques were also produced in 5 to 6 days under an agar overlay when a medium containing fetal calf serum was employed. Kinetics of plaque formation were also studied, and it was shown that fowlpox virus plaque diameters grow at a linear rate.

Langat Virus

Two plaque-purified strains of Langat virus were studied to find markers that would distinguish strains of reduced pathogenicity suitable for vaccines. The following properties were examined: 1) pathogenicity in mice, 2) plaque morphology in chick-embryo cell cultures and the course of multiplication and antigen production, 3) thermal-inactivation rates, 4) chromatography on calcium phosphate columns, and 6) antigenic differences shown with the modified hemagglutination-inhibition (HI) test of Casals. The plaque assay, with the serum-free agar-overlay medium, was found to be as sensitive as the titration of the infectiousness of virus in mice, but plaque morphology was much the same for the two viruses. The two strains could, however, be differentiated on the basis of degree of peripheral pathogenicity in mice, thermal-inactivation rates, and difference in elution patterns and in antigenicity demonstrable by the modified HI test. The markers presently known still appear to be inadequate for the definition of Langat strains as suitable candidates for live vaccines against the tick-borne complex of group B arboviruses.

Rous sarcoma virus production in mixed cultures of mammalian Rous sarcoma cells and chick embryo cells.

AbstractThe induction of Rous sarcoma virus (RSV) production was analyzed in mixed cultures of mouse or hamster Rous sarcoma cells and chick embryo cells treated with ultraviolet inactivated hemagglutinating virus of Japan (UV‐HVJ). The pre‐treatment of UV‐HVJ with a certain concentration of anti‐HVJ serum increased the yield of infectious RSV in mixed cultures enabling a quantitative analysis of this phenomenon to be made. RSV‐inducing ability of a mixture of HVJ and anti‐HVJ serum was affected by the concentration of anti‐HVJ serum and showed changes in parallel with the cell fusion activity of the mixture. The effect of UV‐HVJ on RSV induction was not achieved by incubation of the cell mixture at 0° C as it was after shaking at 37° C. These findings suggest that the production of infectious RSV in mixed cultures may be initiated in heterokaryons of mammalian Rous sarcoma cells and chick embryo cells. The number of infective centers in mixed cultures was, however, as small as 1% of that of heterokaryons. It is possible that only a small fraction of the heterokaryon population is productive of infectious RSV.

Cells Persistently Infected with Newcastle Disease Virus

The strain of Newcastle disease virus (NDV(pi)) present in persistently infected L cells differed markedly from the Herts strain (NDV(0)) used to initiate the infection. NDV(pi) produced small plaques (less than 1 mm) in chick embryo cell cultures, whereas the wild type (NDV(0)) produced large plaques (2 to 3 mm). The two viruses differed in a number of additional properties. Whereas 80% of adsorbed NDV(0) eluted from chicken red blood cells at 37 C, only about 20% of NDV(pi) was recovered under similar conditions. There was no significant difference in the neuraminidase content of the two viruses. The infectivity of NDV(0) was stable for 1 hr at 48 C, whereas 99.9% of the infectivity of NDV(pi) was destroyed. The two viruses also differed in lethality for chick embryos; NDV(pi) had significantly reduced lethality for 9-day-old chick embryos when compared to NDV(0). In contrast to NDV(0), which produced an abortive infection in L cells, NDV(pi) not only replicated effectively and destroyed these cells, but also induced significantly higher quantities of interferon than did NDV(0). These data furnished additional evidence for the lack of relationship of interferon production to abortive infection of L cells with NDV(0). In contrast, interferon was found to play a significant role in the maintenance of persistent infection.

Two variants of Sindbis virus which differ in interferon induction and serum clearance. I. The phenomenon.

It is a fairly common experience in laboratories using the standard AR-339 strain of Sindbis virus to observe variations in the plaque size of this virus [1-4]. The sizes range from large to small, and include in some cases intermediate plaque variants [3] and an unusually large plaque (U) obtained after cloning of a parent small plaque variant at 40 C [4]. We have used Sindbis virus extensively as an interferon inducer in cell cultures [5-7] and in [8]; that is, in the production of serum interferon shortly after a large dose of virus is inoculated intravenously in rabbits and before the virus replicates to any significant extent [9-11]. In the course of using Sindbis virus, we have noted occasional large plaques (^5 mm) among the usual smaller plaques (^2 mm) in chick embryo cell cultures under Bacto agar (Difco). Recently, to study how variants of 1 virus may differ in the systemic induction of interferon [12], we decided to study the systemic interferon-inducing capacity of small plaque (SP) and large plaque (LP) variants of Sindbis virus. Preliminary results revealed not only that the LP variant induced more serum interferon in animals, but also that it was cleared from the blood stream

Cytotoxicity of filtrates of haemolytic Escherichia coli.

SUMMARY The diffusible haemolysin of Escherichia coli is highly cytotoxic to chick embryo cell cultures, but has no effect on monkey kidney and mouse embryo cell cultures. The cytotoxic effects are specific, as they can be neutralised by specific antisera. Culture fluids from non-haemolytic strains of E. coli are non-cytotoxic. The haemolytic activity is thermolabile, but cytotoxicity is thermostable. The antibodies prepared against heated or unheated haemolysins prevent cytotoxicity, but haemolytic activity is neutralised only by antisera prepared against unheated haemolysin. Fraction A of haemolysin is non-haemolytic and non-cytotoxic, whereas fraction B is non-haemolytic but is cytotoxic. The haemolytic and cytotoxic factors of Escherichia coli appear, therefore, not to be identical.

Effect of interferon on Sindbis virus growth in chick embryo cell cultures.

A study of the effect of interferon on the growth of Sindbis virus in cultures of chick-embryo cells has shown that interferon forms an association with cells (uptake) and that the action of interferon is concentration rather than amount dependent. Evidence has also been obtained that interferon acts to reduce the yield of virus from cells, but does not reduce the number of cells synthesizing virus (all or none effect).

Properties of the Attenuated Vaccine Strain of Duck Hepatitis Virus

The attenuated vaccine strain of duck hepatitis virus (DHV) grew to titers of 107.5 to 108.5 ELDs0 (egg-lethal doses)/0.1 ml when grown in 10-day-old embryonated chicken eggs and would grow to titers of 104 to 104-5 ELDso in trypsin-dispersed primary chick embryo cell culture. DHV was shown to have the following properties: RNA, 50 mt in size, chloroform-resistant, pH-3.0-resistant, trypsinresistant, relatively formalin-sensitive, heat-stable (50 C) and no effect of 1M divalent cation (Mg++) upon heat stability. Having these properties, DHV would best classify as a duck picornavirus.

A factor capable of enhancing interferon synthesis

In chick embryo cells aged for several days in vitro, interferon synthesis induced by ultraviolet-irradiated Newcastle disease virus occurs faster and reaches a higher maximum level than in 1-day-old cultures. The enhanced capacity to produce interferon appears to be mediated by a factor produced in aging cultures of chick embryo cells and released into the medium. The factor exhibits properties of a protein.

Biological Markers for Differentiation of Herpes-virus Strains of Oral and Genital Origin

Summary Herpes simplex virus from mouth and genitalia could be distinguished antigenically and also differed in certain biological properties. Viruses isolated from oral lesions replicated better in rabbit kidney cell cultures than did viruses isolated from genital lesions; however, the total number of virus particles produced was the same for both virus types. A greater proportion of particles in stocks of the oral strains grown in rabbit kidney cells were enveloped than in stocks of genital strains grown in the same cells. The genital strains formed plaques in chick embryo cell cultures, but the oral ones did not and replicated poorly. The genital strains were more neurovirulent when injected intracranially into mice than were the oral strains, based on the number of p.f.u. injected; however, there was no difference in neurovirulence if the doses were calculated in terms of total particles injected. These differences provide useful markers for characterizing the types of herpes virus.