An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers

Summary Various factors were examined for their influences upon the plaque assay of herpes simplex virus in chick embryo monolayers. The factors analysed included the process of monolayer preparation, constituents of growth medium, conditions of virus adsorption and conditions of maintenance of cells after infection, and eventually a standard procedure was established. An important difference from the conventional method was the use of a small inoculum size, based on the fact that when the inoculum size was 0.05 ml. per 60-mm dish a higher titer was obtained than when larger inocula were used. Virus adsorption was practically over in one hour at 37° C leaving approximately 30% of the inoculated virus unadsorbed. This adsorption rate could not be improved even when adsorption was allowed to proceed in a heavy cell suspension. The titration efficiency as well as the plaque size was greatly influenced by the passage history of the virus. An HF strain which had gone through numerous egg passages showed equal plaque and pock titers but serial passage in chick embryo fibroblasts resulted in a ten times higher plaque titer than the pock titer. Similar trends appeared also with other less egg-adapted strains. The present chick embryo plaque assay was much more sensitive thanRussel'a plaque assay in HeLa cells even for a highly HeLa-adapted subline of the HF strain. The possibility that one chick embryo plaque-forming unit of the HF strain represents close to one infectious virus particle is discussed.The present work was aided in part by Research Grant GA MNS 61168 supplied from the Rockefeller Foundation, U. S. A.     

Effect of alkaline maintenance medium upon the growth of rabies virus in chick embryo cells

HEP Flury strain of rabies virus was propagated in chick embryo cells under maintenance media of different pH. It was found that viral growth was better and reached a markedly higher maximum titer when the initial pH of maintenance medium was 8.2 to 9.0 than when it was 7.4. The enhancement of viral growth was not ascribable to mere neutralization of acids produced from infected cells, because the different media became almost equally neutral within an early phase of growth curve. Serial passage of the virus in chick embryo cells using pH 8.2 maintenance medium resulted in altered growth characteristics of the progeny virus; first, the virus so passaged could now grow equally well under alkaline and neutral maintenance media, and, secondly, autointerference observable with the parent virus eventually lowered virus yield when neutral maintenance medium was used, but this effect of undiluted passage was eliminated by the use of pH 8.2 maintenance medium.     

An electron microscopic study of the surface structures and hemadsorption on chick embryo cells infected with rabies virus

Summary Characteristic alterations at the surface of chick embryo cells infected with the HF-TC strain of rabies virus and the binding sites of hemadsorption were studied employing both scanning and transmission electron microscopes. The initial alteration of the cell surface structure revealed by scanning electron microscopy was an appearance of elongated and reticulated microvilli on the 2nd day after virus inoculation. On the 3rd day, numerous bullet-shaped virions could be seen budding as single, tetrapod-like structures and as radial projections both from the perikarya and microvilli. Thereafter, elongation of microvilli, formation of numerous blebs in various sizes, disappearance of filopodia, and rounding up of infected cells were observed as characteristic cytopathic effects by rabies virus infection.The attachments of goose erythrocytes to the infected cells occured in two forms. The one was adsorption of erythrocytes to the cell surface involving microvilli and filopodia in the absence of detectable virus, and the other was adsorption of erythrocytes to the virus particles budding from cell surface. The former could be seen from the early stage of infection through the end of observation period, while the latter was observed only on and after the 3rd day after virus inoculation. These findings were also confirmed with transmission electron microscopy.With 10 Figures     

A sensitive plaque assay for influenza virus A2 Japan in chick embryo fibroblast cultures

Summary Growth of influenza virus type A₂, strain Japan/305/57 (formerly grown in the allantoic cavity of 11-day old chick embryos), was obtained in cultures of chick embryo fibroblasts. Virus, passed four times in chick embryo fibroblasts, followed by one passage in the allantoic sac of 11-day old embryonated eggs, gave clear plaques in a chick embryo monolayer under a serum-free agar overlay with an added DEAE-dextran concentration of 0.5 mg./ml. After three days incubation at 37° C in stoppered flasks the plaques reached a diameter of 2–3 mm. An approximately straight-line dose-response relationship was observed between the square root of the number of plaques produced and the relative concentration of virus. Specificity was demonstrated by quantitative neutralization with immune serum.     

Electron microscopy of Lednice virus in chick embryo cells

Replication of Lednice virus in chick embryo cells was studied for 72 hr after inoculation by infectivity titration and the indirect immunofluorescence technique. At 24 and 48 hr after inoculation, electron microscopy revealed spherical virions of uniform morphology, 80-105 nm in diameter, which were localized mostly extracellularly.     

An improved plaque assay for varicella virus

Summary Varicella virus was found to form plaques more clearly defined and about twice greater in number at 31° C than 37° C in human embryonic lung fibroblasts, and an improved method of plaque assay for the virus was developed.With 1 Figure     

Polypeptides induced in chick embryo cells by Kemerovo virus

Fifteen polypeptides induced by Kemerovo virus were detected in chick embryo cells (Mr 140, 98, 89, 72, 65, 62, 57, 54, 50, 47, 43, 41, 39, 31 kD, and 30 kD). Nine of them, namely the 140, 98, 65, 62, 57, 54, 50, 47 kD, and 41 kD polypeptides were also found in the partially purified virus. However, the latter contained also considerable amount of host cell proteins, predominantly the 205 kD, 45 kD, and 37 kD polypeptides. In the electron microscope the spherical viral particles exhibited a poorly defined surface structure of a diameter of 70-75 nm.     

Synchronization of Rous sarcoma virus production in chick embryo cells

The growth of chick embryo fibroblasts transformed by and producing the Schmidt-Ruppin strain of Rous sarcoma virus was synchronized by exposure to serum-free medium. It was found that virus-specific RNA and protein synthesis and virus release are synchronized in these cells and occur in the early S and G₁ phases of the cell cycle, respectively. These findings support the hypothesis that RSV synthesis is dependent upon specific host cell processes. The duration of the phases of the cell cycle in transformed cells could not be distinguished from the pattern in uninfected cells. Evidence is presented which indicates that the synchrony of transformed cells is achieved by the low pH of the serum-free medium rather than by the absence of serum.     

Rapid cytopathic effect with rabies virus in fused hamster embryo cells

Summary A line of hamster embryo cells fused with inactivated Sendai virus was infected with a strain of fixed rabies virus. The growth of rabies virus in such a system produced a characteristic cytopathic effect.Work done while the senior author was on leave from the Panamerican Zoonosis Center, Casilla de Correos 23, Ramos Mejía, Buenos Aires, Argentina.