Allantoic

Fig. 8. Preparative isolation of hexon antigen of EDS-76 by hydrophobic-interaction chromatography on Butyl-PG column (2x5 cm) (A) application of the allantoic fluid diluted (1 5) by 50 mM potassium acetate, pH 4,130 ml (B)0.01 mol/1 potassium acetate, pH 5.5 (C) 0.01 mol/1 potassium bicarbonate pH 8.0, 10% isopropanol (D) 0.01 mol/1 potassium carbonate pH 9.6, 10% isopropanol. EDS-0 — components of alantoic fluid eluted with buffer A, EDS-1 — desorbed hexon fraction eluted with buffer C, EDS-2 — fraction desorbed with buffer D [56]...

Embryonated hens eggs are still the most convenient hosts for the growth of the vimses that are needed for influenza and yellow fever vaccines. Influenza vimses accumulate in high litre in the allantoic fluid of the eggs and yellow fever vims accumulates in the nervous systems of the embryos. 

Influenza (split virion)t Allantoic fluid from embryonated hens eggs infected with influenza viruses A and B 1 Harvest of viruses 2 Disruption with surface active agent 3 Blending of components of different serotypes Assay of haemagglutinin content by immunodiffusion Inoculation of embryonated hens eggs to exclude live virus... 

Although HIV infection inhibition is mainly due to interaction of fullerene derivatives with viral enzymes, it is necessary to consider that this is not the only exploitable mechanism. In fact, the photodynamic inactivation of influenza vims has also been proposed (Zarubaev et al., 2007). The outer viral membrane is destroyed, while it seems that the protein profile of allantoic fluid, in which the vims was propagated, remains unchanged, confirming one more time the great potentiality of fullerene. 

Zarubaev VV, Belousova IM, Kiselev OI, Piotrovsky LB, Anfimov PM, Krisko TC, Muraviova TD, Rylkov VV, Starodubzev AM, Sirotkin AC (2007) Photodynamic inactivation of influenza virus with fullerene C60 suspension in allantoic fluid. Photodiagn. Photodyn. Ther. 4 31-35. 

The purpose of the present study was to investigate a photodynamic inactivation of influenza vims in the allantoic fluid of chicken embryos. Further, we have evaluated nonspecific effects of photodynamic treatment on the components of biological fluids. 

In a separate set of experiments, in order to remove soluble components of allantoic fluid, the fluid was centrifuged for 1 h at 200,000g. Supernatant was removed and virus was resuspended in saline in a volume equal to the initial volume of allantoic fluid. 

A confluent monolayer of Madin-Darby canine kidney (MDCK) cells was grown in 96-well plates. Serial tenfold dilutions in minimal essential medium were prepared from the aliquots of allantoic fluid taken from the irradiated specimen. These dilutions were applied to MDCK cells and incubated for 48 h at 36 °C in 5% C02. The cells were then washed two times for 5 min with phosphate buffered saline (PBS) and incubated for 1 h with 100 pi of 0.5 mg/ml solution of 3-(4,5-dimethyl-thiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT, ICN Biochemicals Inc., Aurora, Ohio). After lh, the colored deposit was dissolved in 100 pi DMSO, and optical density in the wells was measured on plate reader Victor 1420 (Perkin Elmer, Finland). Based on the data obtained, the infectious titer of the vims was determined as a decimal logarithm of reciprocal to the dilution of the specimen causing destruction of 50% of cells. The inhibiting action of irradiation was evaluated by decreasing the vims titer. 

Sample preparation. All allantoic fluid of chicken embryos or calf serum used in experiments contained influenza virus (104—106 EID50/ml). The samples of biological fluids underwent photodynamic treatment as described above. One milliliter aliquots were taken before treatment and at 3 and 6 h after the start of experiment. To analyze the effect of photodynamic treatment on proteins we used alkaline denaturing electrophoresis in the presence of sodium dodecylsulfate (SDS) and P-mercaptoethanol (P-ME). 

Separation of proteins. Aliquots of biological fluids (blood serum or allantoic fluid) were mixed with the Laemmli buffer for the samples. For proteins denaturation samples were boiled for 5 min. Samples were loaded in and separated in the gel by Miniprotean II unit (BioRad). The voltage was 80 V when samples were in concentrating gel and 100 V when in separating gel. The movement of the proteins was tracked by bromophenol blue. 

The infectious titers of influenza vims were determined in the irradiated specimen at different time points. The kinetics of the vims activity in saline and allantoic fluid in the presence of fullerene and oxygen are presented in Fig. 5.1. 

As can be seen from the data presented, the suspension of crystal C60 did not itself affect the influenza vims. In all control experiments without oxygen and irradiation the infectious activity of the vims remained on the same level throughout the experiment (Fig. 5.1). This was true for both vims in original allantoic fluid and vims resuspended in saline. Exposure of the vims to oxygen without irradiation did not affect the viral titer either. 

When exposure to oxygen and light irradiation were commenced simultaneously, the infectious activity of the vims remained on the initial level for 30-50 min. This period of induction was observed both in the allantoic fluid and saline. 

Fig. 5.1 Inactivation of influenza virus in allantoic fluid by fullerene. 1 virus in allantoic fluid, no illumination 2 virus in saline, specific illumination 85mW/cm3 and 3 virus in allantoic fluid, specific illumination 85 mW/cm3. The moment of addition of fresh fullerene suspension is indicated by an arrow...

Fig. 5.2 Protein conglomerates in allantoic fluid after 5 h of irradiation in the presence of Cm and oxygen. Bar lOOnm. Negative contrast...

Fig. 5.3 Destruction of virions of influenza under irradiation in the presence of suspension and Or (a) Intact virions in allantoic fluid and (b) virions after 5h of irradiation in presence of oxygen. Note the fragmentation of the outer membrane of the virions and the loss of a significant part of surface glycoproteins. Negative contrast, bar lOOnm...

Results of electrophoretic analysis of proteins in the allantoic fluid and blood serum before and after photodynamic treatment are presented in Figs. 5.5 and 5.6. Visually, we did not detect any changes in the position and intensity of protein bands. In order to quantitatively analyze these parameters we scanned the gels and measured the relative optical density of the bands (Figs. 5.7 and 5.8). 

Fig. 5.5 Electrophoresis pattern of proteins in the allantoic fluid of chicken embryos. Lanes 1-3 intact fluid lanes 4-6 fluid after 6h of irradiation...

Fig. 5.7 Photometric representation of electrophoresis results. Allantoic fluid before (red) and after (blue) 6h of irradiation (See Color Plates)... 

In the present study we have investigated the process of photodynamic inactivation of influenza vims in the allantoic fluid of chicken embryos. This inactivation has been realized by C60 water suspension used as a photosensitizer. Similar studies have been carried out previously by Kaserman and Kempf (1997, 1998). Unlike the latter studies, in which viruses were inactivated in salt solutions (buffer), our experiments were performed in a natural biological fluid that contains all typical components (proteins, lipids, salts, etc.). Comparing the viral inactivation over time in our experiments with previous results we conclude that the process described by Kaserman and Kempf (1997, 1998) was more time-consuming, a fact that may significantly restrict its practical use. 

The course of viral photodynamic inactivation in allantoic fluid is probably directed by the following process the initial period of induction is a result of the quenching of singlet oxygen (or other reactive forms of oxygen) by natural antioxidants present in the fluid (Kolb, 1991). Subsequently, a dramatic drop in the viral titer was observed. This then reached a plateau due to the consumption of the active fraction of the fullerene. This may be indirectly confirmed by the results of viral titration after the addition of a new portion of fullerene when the process of inactivation was observed again. 

---