Aujeszky’s disease virus production in disposable bioreactor

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 10⁶ cells ml^-1, the reactor was inoculated with 9 ml of gE^- Bartha K-61 strain ADV suspension (10^5.9 TCID₅₀) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10^7.0 TCID₅₀ ml⁻¹, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.     

Uptake of tritiated thymidine in muscle of juvenile carp

In juvenile carp (4.5–6 cm s.l .) labelled myosatellite cell nuclei are found 24 h after injection of tritiated thymidine, but labelled myonuclei after 48 h, indicating that fish myonuclei do not proliferate but originate from undifferentiated myogenic cells. The time pattern accords with the estimated cell-cycle time of adult myogenic cells of carp.     

Permeability changes resulting from virus-cell fusion: temperature-dependence of the contributing processes

Summary A new assay for membrane fusion, using the fluorescent probe pyrene-sulphonyl-phosphatidyl ethanolamine, has been developed. Fusion between the envelope of Sendai virus and human erythrocytes or Lettre cells has a Q₁₀ of 4 at 37° C, increasing to 7 at 7 ° C; there is no lag to onset of fusion. Viral neuraminidase has a Q₁₀ of 2.3 between 37° C and 4° C. Its action limits the extent of fusion by causing the elution of virus; this effect is particularly marked at low temperature because of the difference in Q₁₀ for fusion and neuraminidase. The temperature-dependence of the initiation of permeability changes following the removal of inhibitory amounts of Ca²⁺ is 2; thus membrane fusion is the principal temperature-sensitive step during the permeabilization of cells by Sendai virus. A recovery process, by which cells become insensitive to the removal of Ca²⁺ and which therefore limits the extent of permeabilization, has a Q₁₀ of 7.4 between 37° C and 21° C. It is concluded that the lag to onset of permeability changes is not due to a lag in virus-cell membrane fusion, but to the gradual acquisition of a threshold level of membrane damage; the extent of permeabilization depends on the rate of fusion relative to the rates of neuraminidase and recovery.     

Difference in capacities for virion-to-virion fusion of young and aged HVJ (Sendai virus): A model of membrane fusion

Summary Young and aged HVJ virions differ structurally and morphologically due to changes that occur during aging in vitro or in ovo. Young virions soon after their budding off are rodshaped, rigid and relatively uniform in size, whereas virions that have aged in vitro after their formation are round, nonrigid and variable in size. These changes during aging seem to be due to the variation of M protein, a skeletal protein that is associated with both the envelope membrane proteins and nucleocapsid strands in the virions. The capacities for virion-to-virion fusion of young and aged virions were compared to clarify the relation between the membrane fusion and membrane-associating skeletal proteins. On treatment with polyethylene glycol (PEG), aged virions readily fused, forming large virion vesicles, but young virions were resistant to fusion. Further, aged virions fused even on incubation at 37°C without the fusogen. Thus the capacity for virion-to-virion fusion evidently increases during aging of virions. This result suggests that skeletal proteins associating with the biological membrane are important for preventing membrane fusion, and that virion-to-virion fusion is a good model system for use in studies on the mechanism of membrane fusion.     

Inhibition of virus-induced plaque formation by atoxic derivatives of purified cobra neutrotoxins

Venom from Naja naja siamensis was resolved into 16 toxic and nontoxic fractions by chromatography on SP-Sephadex, C-25. The principal neurotoxin preparations were chromatographically and electrophoretically homogeneous.Of the purified constituents, only the principal neurotoxin and minor neurotoxins were precursors of inhibitors of plaque formation among baby hamster kidney fibroblasts infected with Semliki Forest Virus.     

Isolation of 3-(4,8,12-Trimethyltridecyl)-furan (“Phytofuran”) from Burley Tobacco

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.     

原代地鼠肾细胞及狂犬病毒不同培养方式的研究

目的通过比较原代地鼠肾细胞在转瓶、微载体、细胞工厂的3种培养方式的培养效果,为原代地鼠肾细胞选择一种易扩大规模、培养高质量细胞的培养方式,进而提高狂犬病毒的产量。方法消化取得的细胞悬液分别在转瓶、微载体、细胞工厂中培养,通过显微镜观察细胞形态、计数等结果比对培养的差别。结果细胞工厂可以很好地培养原代地鼠肾细胞和狂犬病毒;而细胞在微载体上贴附性差,生长不好。结论实验结果表明细胞工厂可以取代转瓶,用于大规模培养原代地鼠肾细胞扩大狂犬病毒的产量。     

Use of a fluorescence assay to monitor the kinetics of fusion between erythrocyte ghosts,as induced by sendai virus

The kinetics of the fusion process between erythrocyte ghosts, as induced by Sendal virus, were readily revealed by a simple fluorescence procedure previously employed to characterize the fusion of viruses with biological membranes. The method relies on the relief of fluorescence selfquenching of the membrane-inserted probe octadecyl Rhodamine B chloride (R₁₈) as occurs when labeled membranes fuse with unlabeled counterparts. The kinetics of R₁₈ insertion into ghost membranes, the non-exchangeable properties of the fluorophore and the kinetics, and some characteristics of Sendai virus-induced fusion of ghosts, are described. We propose that the experimental approach may be particularly advantageous to obtain insight into the efficiency and mechanism of a wide range of fusogens, capable of inducing fusion of erythrocyte membranes.     

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Summary Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. Within 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed. This work was supported in part by Public Health Service Research Contract FDA 233-74-1035 and by Research Grant AI-08648 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

Gating Movements of Colicin A and Colicin Ia Are Different

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20–22°C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10–40°C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.This revised version was published online in August 2005 with a corrected cover date.     

Role of hydrophobic interactions in the fusion activity of influenza and sendai viruses towards model membranes

We have studied the role of hydrophobic interactions in the fusion activity of two lipid enveloped viruses, influenza and Sendai. Using the fluorescent probe ANS (1-aminonaphtalene-8-sulfonate) we have shown that low-pH-dependent influenza virus activation involves a marked increase in the viral envelope hydrophobicity. The effect of dehydrating agents on the fusion activity of both viruses towards model lipid membranes was studied using a fluorescence dequenching assay. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced the initial rate of the fusion process, the effect of dimethylsulfone doubling that of dimethylsulfoxide. The effect of poly(ethylene glycol) on the fusion process was found to be dependent on the polymer concentration and molecular weight. In general, similar observations were made for both viruses. These results stress the importance of dehydration and hydrophobic interactions in the fusion activity of influenza and Sendai viruses, and show that these factors may be generally involved in membrane fusion events mediated by many other lipid enveloped viruses.