Chick Embryo Fibroblast Monolayers Research Articles

Vergleichende Untersuchungen an Paramyxovirus‐Isolaten aus Wildvögeln*

As a result of a surveillance programme in North-Germany, paramyxovirus-isolates of serogroup 1 with different pathogenicity were isolated from different species of feral birds (Black-headed gull, mallard, tawny owl, tree sparrow, mute swan). In chick embryo fibroblast monolayers these isolates produced plaques exhibiting different plaque morphology. After cloning the isolates, further physicochemical, serological and pathogenicity testings were performed. On the basis of mean death time and intracerebral pathogenicity index, two of the isolates were found to be avirulent (lentogenic), whereas the others proved to be virulent (velogenic). SDS-Polyacrylamide-gel-electrophoresis showed polypeptide patterns similar to that of the PMV-1 reference strains. Summarizing all relevant findings of these investigations under consideration of the suggestions for the virological description of avian PMV (Alexander, 1987; Alexander et al., 1987; Who-Expert-Committee, 1980), including the recently established serotyping by means of monoclonal antibodies, the following designations of the PMV isolates tested herein are proposed: 1. PMV-1/black-headed gull/Germany/SSP-233/83, lentogenic NDV, monoclonal antibody group G 2. PMV-1/mallard/Germany/SSP-284/83, lentogenic NDV, monoclonal antibody group H 3. PMV-1/tawny owl/Germany/SSP-262/84, velogenic NDV, monoclonal antibody group B 4. PMV-1/tree sparrow nestling/Germany/SSP-352/84, velogenic NDV, monoclonal antibody group B 5. PMV-1/mute swan/Germany/SSP-204/85, velogenic NDV, monoclonal antibody group B.

A comparison of three methods for detecting antibodies to turkey rhinotracheitis virus

Sera from a flock of naturally infected commercial turkeys were tested for antibodies against turkey rhinotracheitis virus using an indirect immunofluorescence test with infected turkey embryo tracheal organ cultures, a serum neutralisation test using chick embryo fibro-blasts or liver cells and an ELISA. Antibodies were detected by all tests within 5 days of the appearance of clinical respiratory disease. Serum neutralising antibodies detected in chick embryo fibroblasts rapidly achieved their peak titres and were decreasing by day 13. ELISA antibodies peaked on day 13. On days 13 and 34 significant correlations were obtained for immunofluorescence and ELISA and for ELISA and microneutralisation in fibroblasts. For both the latter tests there was a good correlation between the results obtained for individual birds within the flock on days 13 and 34. ELISA and serum neutralisation in chick embryo fibroblast monolayers would appear to be sensitive and reliable tests for detecting circulating antibodies against turkey rhinotracheitis virus in commercial flocks.

The genome of incomplete influenza virus

After three high-multiplicity passages of the WSN strain of influenza virus in primary chick embryo fibroblast monolayers, the virus produced contained much less of the largest RNA segment and reduced amounts of the next two largest segments as compared with normal virus. The double-stranded RNA isolated from the cells which produced these particles also showed a similar decrease in these three segments as did the cRNA, both that associated with polysomes and non-polysome associated. Virions lacking these RNAs did not transcribe these segments in vitro but did transcribe the other five segments. An analysis of the molar ratios of segments indicated that the three largest segments are the most seriously affected. There was no evidence for random loss of segments either by these analyses or by our inability to enhance infectivity by clumping defective virions.

Morphologic heterogeneity in egg- and monolayer-propagated Newcastle disease virus

Newcastle disease virus (NDV) propagated in monolayers of chick embryo fibroblasts (M-NDV) has been found to differ morphologically from NDV propagated in embryonated eggs (E-NDV). Virions of M-NDV sediment faster, are lower in density, are about four times larger, and are more fragile to degradative forces encountered during high speed pelleting and rebanding in CsCl than E-NDV virions. These differences have been shown to be host controlled and are not due to contaminant virus, differences in growth medium, virion clumping, or time of harvest. Although both E- and M-NDV populations contain multiploid virions, these virions do not exhibit multihit inactivation kinetics. The possible genetic consequences of the differences between E-NDV and M-NDV are discussed.

The inhibition of influenza virus RNA synthesis by actinomycin D and cycloheximide

Chick embryo fibroblast monolayers infected with influenza virus (WSN strain) were treated with either actinomycin D or cycloheximide at various times after infection. Analysis of cell homogenates indicated that in the presence of actinomycin D, synthesis of RNA complementary to virion type RNA was preferentially inhibited. Consequently, no labeled polysomes are found in cells so treated, although virion-type RNA, isolated as the RNP, was found. Cycloheximide, on the other hand, while suppressing the synthesis of complementary type RNA, completely inhibited the synthesis of virion-type RNA. These results confirm and extend the work of Scholtissek and Rott (1970) that showed the same differential effect of these two drugs on RNA synthesis and are added evidence that in influenza virus-infected cells the messenger RNA is that RNA which is complementary to the RNA found in virions.

Comparative Studies on Large- and Small-Plaque-Forming Clones of Newcastle Disease Virus (Miyadera Strain)

The Miyadera strain of Newcastle disease virus (NDV) consisted predominantly of virus particles forming small plaques on monolayers of chick embryo fibroblasts (CEF), and contained small amounts of virus particles forming large plaques. These large- and small-plaque-forming clones of this virus (NDV-L and NDV-S) were isolated. The small size of the NDV-S plaques did not appear to be due to an agar inhibitor. NDV-L produced a much higher yield of infective virus particles in CEF and they were released more completely from the infected cells than were those produced by NDV-S. The yield of infective virus of NDV-L per cell from cultures of CEF was comparable to the yield from the allantoic cells. The infectivity/hemagglutinin ratio for NDV-L from CEF was as high as the ratio for virus from the allantoic cells, but the ratio for NDV-S from CEF was lower. NDV-S demonstrated an autointerference phenomenon in CEF when infected at high multiplicities, but NDV-L did not. Contrary to virus multiplication, NDV-S exhibited a more rapid and marked cytopathic effect on monolayers of CEF than NDV-L. In the allantoic cavity of eggs NDV-S produced slightly higher virus yields than NDV-L. No correlation existed between plaque size of the two viruses and the capacity to induce interferon synthesis or the susceptibility to the action of interferon. The properties of both distinctive plaque isolates were stable on egg passage.

Enhancement of Sindbis virus infectivity by reduced salt concentration.

The efficiency of plaquing (EOP) on chick embryo fibroblast monolayers of three temperature-sensitive mutants of Sindbis virus defective in late functions was enhanced 10- to 11-fold when the inoculum was applied in medium containing 66% of the normal concentration of sodium chloride. Studies with one of these mutants revealed similar enhancement over a broad range of reduced salt concentrations. The EOP of wild-type virus and of a temperature-sensitive mutant defective in RNA synthesis were also enhanced, but to a lesser degree. No increase in EOP occurred if the exposure of virus to reduced salt concentration ended before the virus suspension was applied to the monolayers; thus, a virus-cell interaction appeared to be involved. The salt-sensitive step occurred within the first hr of infection, but subsequent to virus adsorption.

Immunization against Marek's Disease

The turkey herpesvirus (HVT) and Marek's disease (MDHV) herpesvirus produce a similar virus-like cytopathic effect in monolayers of chick embryo fibroblasts. Discrete foci of rounded refractile cells and syncytial masses with some of the nuclei containing Cowdry type A inclusions were observed in affected monolayers. Electron micrographs indicate that both isolates are herpesviruses. The HVT appears to differ from MDHV in that the MDHV fluorescent antibody conjugate does not stain monolayers infected with HVT, nor does the MDHV antigen react with HVT antisera in the agar gel precipitin test. Continuous passage of MDHV in cell culture resulted in the loss of its oncogenic potential for the chicken, whereas HVT was found not to be oncogenic for chickens even with low cell-culture passages. One-day-old chicks vaccinated subcutaneously with either cell-free HVT, suspension of HVT in cell culture or attenuated MDHV were protected against MD when immunity was challenged by natural or artificial exposure. Lyophilized HVT or MDHV vaccines were found to be less effective than fresh live-virus vaccines. Investigations on the route of exposure indicate that these vaccines are most effective by the parenteral inoculation of these materials.

Effect of interferon on the replication of Sendai virus.

SUMMARY The incorporation of [3H]uridine into the four species of RNA specified by Sendai virus in monolayers of chick embryo fibroblasts was inhibited to an equal degree by addition of purified chick interferon to cultures before infection with virus. The inhibition of RNA synthesis was dose-dependent. Similarly, [3H]uridine incorporation into RNA of virus nucleocapsid and polyribosomes was completely inhibited by pretreatment with interferon. When interferon was added 2 hr after infection, there were only small effects on the synthesis of total virus-specific RNA and significantly greater reduction was observed in [3H]uridine incorporation into the RNA of nucleocapsid and polyribosomes. Although Sendai infection does not interfere with host-cell protein or RNA synthesis, interferon added 6 hr or later after infection did not affect any Sendai replicative functions.

Comparative studies on development of rabies virus in different host cells

The HEP Flury strain of rabies virus, which had been adapted to chick embryo fibroblast monolayers, was utilized for comparative studies of viral morphogenesis in cultured cells and brain tissues. It was found that, in the former case, numerous viruses were formed by a budding process from the cell surface, whereas it was completely restricted to the intracytoplasmic membranes closely associated with the characteristic matrix in neurons. These findings suggest that the site of the rabies virus assembly is not exclusively within the intracytoplasmic membranous system, but varies, depending on different combinations of virus and host cell. It was noted that the virulence as well as cytopathological change in baby mice did not vary through the 27th-passage level in chick embryo fibroblasts.

Histone-mediated clumping of Newcastle disease virus (NDV)

Newcastle disease virus (NDV) propagated in monolayers of chick embryo fibroblasts (M-NDV) has been found to differ morphologically from NDV propagated in embryonated eggs (E-NDV). Virions of M-NDV sediment faster, are lower in density, are about four times larger, and are more fragile to degradative forces encountered during high speed pelleting and rebanding in CsCl than E-NDV virions. These differences have been shown to be host controlled and are not due to contaminant virus, differences in growth medium, virion clumping, or time of harvest. Although both E- and M-NDV populations contain multiploid virions, these virions do not exhibit multihit inactivation kinetics. The possible genetic consequences of the differences between E-NDV and M-NDV are discussed.

Sindbis virus infection of chick and hamster cells: Synthesis of virus-specific proteins

In monolayers of chick embryo fibroblasts or BHK cells infected with Sindbis virus in the presence of actinomycin, host protein synthesis is inhibited by at least 80–90% between 2 and 3 hours after infection. Thereafter, Sindbis specified polypeptide chains form 60–70% of total protein synthesis. In particular, synthesis of the membrane protein of the virus constitutes 25% of total protein synthesis. Twelve to 16 polypeptides induced by virus infection have been identified in infected cells by acrylamide gel electrophoresis. The pattern of virus-specified proteins observed depends upon the length of the labeling period, the host cell, and mutations in the virus genome. The molecular weight of the membrane protein of the Sindbis virion has been estimated to be 53,000 and the core protein is estimated to be 30,000 in molecular weight.

Degradation of cellular ribonucleic acid in Newcastle disease virus infected cells.

Summary Following Newcastle disease virus infection, the synthesis of both cellular RNA and DNA in chick embryo fibroblast monolayers was inhibited. In such monolayers, Newcastle disease virus infection did not significantly reduce the content of cellular DNA within 10 hr, but did reduce the amount of cellular RNA by about 50% in that time. A part of the RNA was degraded into acid-soluble material and leaked out of the cells. Some fraction of the ribosomal RNA gave rise to small molecular weight fragments which sedimented near the transfer RNA region as determined by sucrose density gradient centrifugation. Evidence is presented indicating that virus-induced proteins, which were synthesized by 6 hr after infection, were involved in the RNA degradation process.

Quantitative haemadsorption. A fast and reliable screening test for anti-myxoviral agents.

Quantitative haemadsorption proved to be a fast and reliable method in antiviral screening, using the haemagglutinating viruses of influenza A2 Japan, influenza A Swine (var), and parainfluenza Sendai. In the case of influenza B Johannesburg the method proved less reproducible. Evaluation of this method for these viruses, using monolayers of chick embryo fibroblasts and calf testicle cells is presented. The haemadsorption development paralleled the formation of haemagglutinin. The relation between dose and effect of 1-adamantanamine hydrochloride when used against the multiplication of influenza virus A Swine (var) is revealed by this method.

Differentiation of Representative Newcastle Disease Virus Strains by Their Plaque-Forming Ability on Monolayers of Chick Embryo Fibroblasts

The 125 isolates of Newcastle disease virus in the Repository at the University of Wisconsin have been recovered from several species of animals and from many countries over the past three decades. This collection provided an opportunity to study the usefulness of plaque morphology as a means of strain identification and to establish the extent to which these isolates were internally heterogeneous. The plaque morphology of 41 NDV strains was examined.

Isolation of Japanese encephalitis virus from mosquitoes by means of the plaque formation method with chick embryo fibroblast monolayers.

Attempts to use the tissue culture method for primary isolations of arboviruses from mosquitoes have been made by several investigators (Welsh et al., 1958; Lennette et al., 1961; Scrivani & Reeves, 1962), indicating that the method may be satisfactory for the surveillance of mosquito infection with certain arboviruses. Porterfield et al. (1960) showed successful isolation of Rift Valley fever virus from mosquito suspensions by plaque formation in chick embryo fibroblast (CEF) monolayers. The CEF is well known to be susceptible also to Japanese encephalitis virus (JEV) .In the present work, an attempt was made to apply the plaque formation method with CEF monolayers to the primary isolation of JEV from field-caught mosquitoes, and the isolation efficiency by this method was evaluated by a simultaneous inoculation into suckling mice.

Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis.

Bolognesi, D. P. (Rensselaer Polytechnic Institute, Troy, N.Y.), and D. E. Wilson. Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis. J. Bacteriol. 91:1896-1901. 1966.-Infection by Newcastle disease virus brings about a rapid and marked inhibition of cell protein synthesis (CPS) in chick embryo fibroblast monolayers. The block to CPS is initiated about 5 hr after infection, and by 9 hr about 85% of the host protein synthesis is shut off. Azauridine (3 mg/ml), a ribonucleic acid (RNA) synthesis inhibitor, prevents the virus-induced inhibition of CPS when added at the time of infection; but it does not prevent the inhibition when added at 3 hr after infection. When puromycin (60 mug/ml), a protein synthesis inhibitor, was added at 3.5 hr after infection, viral RNA was synthesized in normal amounts, but the virus-induced inhibition of CPS was prevented. Actinomycin D added at the time of infection does not, however, prevent the virus-induced inhibition of CPS. The results of these experiments indicate that proteins synthesized during Newcastle disease virus replication are responsible for the inhibition of host-cell protein synthesis. The synthesis of these inhibitory proteins depends on the prior synthesis of viral RNA.

INHIBITORY EFFECT OF MONONUCLEOTIDES ON VIRUS PLAQUE FORMATION.

SummaryAlkaline hydrolysates of yeast ribonucleic acid were found to inhibit markedly plaque formation by vaccinia and chikungunya viruses in chick embryo fibroblast monolayers. Mononucleotides were subsequently implicated and shown to inhibit vaccinia virus plaque formation. Adenosine monophosphate was the most effective of the nucleotides tested in inhibiting vaccinia virus and the inhibition could be reversed with mixtures of the mononucleotides.

PLAQUE ASSAY FOR PSITTACOSIS VIRUS IN MONOLAYERS OF CHICK EMBRYO FIBROBLASTS.

Piraino, F. (City of Milwaukee Health Department, Milwaukee, Wis.), and C. Abel. Plaque assay for psittacosis virus in monolayers of chick embryo fibroblasts. J. Bacteriol. 87:1503-1511. 1964.-A tissue culture system for the assay of 6BC psittacosis virus by plaque production on chick embryo fibroblast cells is described. Experiments are given which characterize the specificity of the reaction and identify factors found to cause variations in plaque counts. The statistical data obtained indicate this assay to be a precise and highly reliable procedure for the detection of 6BC virus and antibody.

An interferon-like substance released from tickborne encephalitis virus-infected chick embryo fibroblast cells.

AN interference phenomenon between tick-borne encephalitis and Western equine encephalomyelitis viruses in chick embryo fibroblast monolayers has been described1. When chick embryo tissue cultures are inoculated with tick-borne encephalitis virus, the virus multiplies readily, but no cytopathic effect appears until the sixth or seventh day of cultivation. If cultures thus infected had been superinfected with a convenient dose of Western equine encephalomyelitis virus, which normally completely destroys the cells within 48–72 hr., the following events could be observed : (1) complete degeneration of cells caused by this virus and, hence, no interference if tick-borne encephalitis virus-infected cultures were challenged with Western equine encephalomyelitis immediately or 24 hr. after primary inoculation; (2) partial interference if cultures were challenged 48 hr. after inoculating them with tick-borne encephalitis virus ; (3) complete suppression of the cytopathic effect caused by Western equine encephalomyelitis virus if challenged 72 hr. after primary inoculation. No interference was established if interfering virus previously neutralized by immune serum was added to cultures.