[Validation of the modified algorithm for predicting host susceptibility to viruses taking into account susceptibility parameters of primary target cell cultures and natural immunity factors].

The paper presents results of testing a modified algorithm for predicting virus ID50 values in a host of interest by extrapolation from a model host taking into account immune neutralizing factors and thermal inactivation of the virus. The method was tested for A/Aichi/2/68 influenza virus in SPF Wistar rats, SPF CD-1 mice and conventional ICR mice. Each species was used as a host of interest while the other two served as model hosts. Primary lung and trachea cells and secretory factors of the rats' airway epithelium were used to measure parameters needed for the purpose of prediction. Predicted ID50 values were not significantly different (p = 0.05) from those experimentally measured in vivo. The study was supported by ISTC/DARPA Agreement 450p.

[Quantitative assessment of in vitro neutralizing activity of secretory factors from rat lungs against influenza virus].

Secretory factors were isolated by lung wash followed by centrifugation to remove cells, dialysis of supernatant to remove NaCl salt, lyophilization of the lavage fluid and resuspention of the lyophilization product in an isotonic NaCl solution. It was shown that biological activity of influenza virus /Aichi/2/68 (3N2) significantly decreased (p = 0,01) from 8,17 +/- 0,10 to 7,14 +/- 0,20 IgEID50/ml during its incubation with secretory factors at 37 degrees C for 1 hr and to 7,92 +/- 0,17 IgEID50/ml in isotonic NaCl solution in the absence of these factors. Their concentration in the incubation medium was estimated to be 9.1 +/- 0.7% of their level in the lungs.

[Comparative analysis of the susceptibility and productivity of respiratory tract target cells of mice and rats exposed to inflienza virus in vitro].

The levels of susceptibility to influenza virus A/Aichi/2/68 H3N2 and the virus yield were determined using primary cells of the trachea and lungs of CD-1 mice and Wistar rats, and for 3 sets of cells obtained from primary lung cells of the both species by centrifugation in the gradient of density and by sedimentation on a surface. The values of ID50 virus dose for 10(6) cells and virus yield per 1 infected cell determined for primary mice cells were 4.0+/-0.47 and 3.2+/-0.27 IgEID50 (lung cells), 3.8+/-0.17 and 3.3+/-0.20 IgEID50 (tracheal cells), and those determined for primary rat cells were 4.0+/-0.35 and 2.1+/-0.24 IgEID50 (lung cells), 3.7+/-0.27 and 2.2+/-0.46 IgEID50 (tracheal cells). The values of ID50 and yield measured for mixtures of cells obtained from primary lung cells by centrifugation in gradient of density and by sedimentation on a surface differed insignificantly (p = 0.05) from the values of the corresponding parameters measured for lung and tracheal cells for both rats and mice. The analysis of data on the variation of the concentrations of different cell types in the experimental cell mixtures shows that type 1 and 2 alveolocytes possess significantly lower (p = 0.05) susceptibility and productivity vs. ciliated cells of the both species. The investigation was conducted within the frame of the ISTC/DARPA#450p project.

[A study of possibility to predict the sensitivity of different respiratory tract divisions to influenza].

To predict a potential value of a viral ID50 for a macro-organism of interest (e.g. humans), it is necessary to determine in vitro two parameters of the interaction of the virus with susceptible cells of the host, i.e. the probability of the virus' productive absorption on a susceptible cell and the average virus yield per cell. A different macroorganism (a model animal) and primary cells obtained from it can be used to determine the value of a scale factor, which accounts for the difference between the values of the probability of the virus' absorption measured in vivo and in vitro. An original mathematical model is used to convert the above-mentioned data to ID50 for the macroorganism of interest. It was shown that the method of cultivating influenza virus (A/ Aichi/2/68) in primary suspension culture of respiratory tract cells of rats and two breeds of mice may be used to estimate potential human susceptibility to novel influenza viruses. This work was sponsored by DAPRA, USA, and performed under the contract 450p to the International Science and Technology Center, Moscow.

[4647-cell culture for preparation of recombinant bivaccine against smallpox and hepatitis B].

The seeding and working banks of a 4647-cell culture have been created. The 4647-cell culture in these banks has a high proliferative activity, as well as the morphology, typical of this line, and the karyotype and the enzymogram, which are characteristic for the cells of an African talapoin (Cercopithecus aethiops). The culture is not contaminated with bacteria, fungi, Mycoplasma, and viruses, including oncoviruses. The deposited 4647 cells have high viral productive properties for the accumulation of the recombinant virus strain b7,5S2-S vaccine and keep the stability of all biological properties during a long-term cultivation. The continuous 4647 cell line was tested at the L. A. Tarasevich State Institute of SK. The seeding and working banks of 4647-cell culture at passages 108 and 128 are recommended as a substrate for cultivation of the strain b7,5S2-S vaccinia, used to prepare a bivaccine against smallpox and hepatitis B.

[Target-cell susceptibility and lung phagocyte activity in mice with influenza virus resistance lowered by glucocorticoidal immunosuppression].

The results of the study showed that subcutaneous kenalog (Kn) lowered the resistance of mice to influenza virus (InV), as was seen by a decrease in 50% lethal dose and an increase in the degree of pulmonary tissue lesion, and the susceptibility of the lungs to InV, seen by the fact that 50% aerogenic infective dose (AID50) was significantly higher in the main group (Kn+InV) than in controls, which received Hanks solution subcutaneously (HS+InV). In vitro, 50% infective doses of InV for suspension of pulmonary and tracheal cells, characterizing their susceptibility to InV, were similar in Kn mice and controls. At the same time, lower resistance and higher degree of pulmonary inflammation noted in Kn mice after receiving a dose of InV that was much higher than an infecting one, was accompanied by the prevalence in the number as well as phagocyte and superoxide-producing activity of neutrophiles (Nph) over the same parameters for alveolar macrophages (AMph) as early as two days after receiving InV dose, vs. InV-infected controls. Evidently, one of the reasons for lower resistance to InV after Kn administration is significant disbalance between the functional activity of AMph and Nph populations. Ineffective AMph clearance of the lungs from InV and excessive number of recruited Nph and products of tissue disintegration may favor the development of respiratory failure and infectious-toxic shock, which leads to lower resistance in animals which receive Kn before InV infection.

[The influence of glucocorticoid immunosuppression on the course of experimental influenza pneumonia].

The study demonstrates the effects of kenalog (Kn), a synthetic glucocorticoid hormone, on the course of virus A/Aichi/2/68 influenza in white mice. In doses of 5 and 10 mg/kg, Kn reduced the weight of the adrenal glands, thymus and spleen, which was accompanied by decrease of the resistance to the mentioned virus, judging by LD50 decrease vs. this index in the control infected group. Besides, four days after infecting with 5 LD50 of influenza virus (IV), lung virus and interferon titers were significantly lower in mice pretreated with Kn vs. mice treated with placebo. Lung cell susceptibility to IV in vitro was identical in mice treated with Kn or placebo. In ultrathin lung sections of IV-infected mice, both experimental and control ones, there was virus budding in bronchial epithelium cells and type I and II alveolocytes. Analysis of inflammatory effusion compound in semithin lung sections 6 days after IV infection, found a substantially smaller number of mature alveolar macrophages (AM) and a bigger number of neutrophiles vs. infected controls. The authors reckon that higher mortality of mice pretreated with Kn before infecting, is caused not by enhancement of IV reproduction in target lung cells during influenza development, but by the contribution of other pathogenic factors. One of those may be increase of neutrophilic migration into the lungs; neutrophiles are more able to realize their significant destructive potential under the condition of reduction in the clearing function of AM and IV infection.

[Clinical trials of oral recombinant bivaccine against variola and hepatitis B during double vaccination].

Clinical trials of oral live recombinant embryonic variola and hepatitis B bivaccine as tablets (Revax-BT) were performed. When volunteers were prevaccinated with oral variola vaccine first in a small dose and, 7, 14, 30, 90, and 180 days later, in a larger dose, a slight reactoginicity was sometimes observed after the first vaccination (with a small dose) whereas revaccination with a larger dose did not give rise to any clinical manifestations. A month after vaccination, a protective level of virus-neutralizing antibodies to vaccinia virus (VV) was observed in 90-100% of the volunteers twice immunized with the bivaccine (in a small dose and in a larger one at an administration intervals of 1-2 weeks under remote revaccination while 6-9 months following vaccination, this level was recorded in 80% of the volunteers. A month following vaccination, 50-55% seroconversion to VV was observed in the volunteers twice immunized with the bivaccine (at an interval of 1 or 3-6 months). Cellular immunity to VV was low (0-20%). Double immunization of volunteers with the oral bivaccine under remote vaccination failed to produce the significant levels of humoral and cellular immune responses to hepatitis B markers. Recombinant VV was not recorded in any blood, saliva, and urine samples taken in the volunteers twice immunized with the bivaccine.

[Preclinical studies of the anticancer adenovirus cancerolysin preparation].

The anticancer drug Cancerolysin has been developed, by using the mutant Adel2 variant of human adenovirus serotype 5 designed at the State Research Center of Virology and Biotechnology. Cancerolysin possesses a high degree of replication activity for complementary cells 293 and p53-deficient tumor cells and, at the same time, has significant replication limitations in normal human cells. Preclinical studies of the drug on laboratory animals (mice, rabbits, guinea pigs) have demonstrated its harmlessness and safety. When stored at -40 and -70 degrees C, the drug showed no significant activity throughout the control observational period (1 year).

[In vitro study of antiviral activity of Myramistin against subtypes H3N2 and H5N1 of influenza virus].

In vitro antiviral effect of myramistin on influenza virus (MDCK cell culture) was studied. The drug showed significant dose-dependent antiviral activity against the virus. When used prophylactically (1 hour before exposure to the virus) in subtoxic doses, myramistin was effective in inhibiting replication of the influenza virus [strains A/Aichi/2/68 (H3N2) and A/Chicken/Suzdalka/Nov-11/2005 (H5N1)]. In urgent prophylaxis (1 hour after exposure to the virus) the protective effect was less pronounced, especially when the contamination dose was high. When the drug was added 12 hours after exposure to the virus, it had no protective effect on the MDSK cell monolayer. The prospects of the myramistin use as a prophylactic agent in grippe are discussed.

Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring.

A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field. The operational principle of the device is based on the passage of air through porous medium immersed in liquid. This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal. As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period. The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses. It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation. Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses. It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate. A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives. The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water. The same trend was observed throughout the entire 4-h experiment. There was no significant difference observed only for the robust vaccinia virus.

[Reactogenicity, safety and immunogenicity of a recombinant bivaccine against smallpox and hepatitis B in limited clinical trials]. / Reaktogennost', bezopasnost' i immunogennost' rekombinantnoi bivaktsiny protiv ospy i gepatita B v ogranichennykh klinicheskikh ispytaniiakh.

The reactogenicity of the embryonic live recombinant variola and hepatitis B bivaccine as tablets (Revax-BT) as well as its safety and immunogenicity were evaluated in clinical trials made in volunteers who had previously immunized or not with variola vaccine. A preliminary conclusion was made on a lack of side effects and drug safety in primary vaccination and been revaccination with low and high doses. Primary immunization of volunteers and as bivaccination with high doses stimulated the most pronounced immune response to the vaccine virus versus such effect observed in immunization of volunteers with low vaccine doses. Humoral immune response to HBs was observed in 75% of volunteers of both groups after as bivaccination. Such response was most pronounced in examinees immunized with low vaccine doses versus those who received high bivaccine doses. At the same time, no protective levels of humoral immunity response to HBs Ag were observed in volunteers first vaccinated.

[A new setup for the generation and studies of mono-disperse microbiological aerosols in medical-and-biological research]. / Novyi kompleks dlia polucheniia i izucheniia monodispersnykh mikrobiologicheskikh aérozolei v mediko-biologicheskikh issledovaniiakh.

A setup for the generation and studies of mono-disperse microbiological aerosols is described in the paper. Coefficients of 3 microm aerosol deposition in the respiratory tract of mice and rats were refined by using the above setup. The probability of deposition of such particles in the trachea and lungs of mice was proven to be equal to 1.2 +/- 0.1% and 2.6 +/- 0.2%, respectively. The probability for rats was equal to 3.2 +/- 0.2 and 11.8 +/- 0.9%, respectively. The distribution of deposited aerosol particles was determined by electron microscopy.

[Pulmonary cell susceptibility in mice and rats to influenza virus when infected in vivo and in vitro]. / Vospriimchivost' kletok legkikh u myshei i krys k virusu grippa pri infitsirovanii in vivo i in vitro.

The purpose of the case study was to evaluate comparatively the relative contribution of cell susceptibility and the inhibiting effect of factors of pulmonary epithelial lining in mice and rats to influenza virus A/Aichi/2/68 (H3N2) adapted to mice as related with the development of infection process in the lungs of experimental animals when infected in vivo and in vitro. Mice and rats were infected aerogenically with different doses of influenza virus. The primary cell-culture suspensions sampled from the lungs of mice and rats were used to study the adsorption and dynamics of influenza virus production in infection by different dose of influenza virus in vitro. The cell suspensions were shown to be able to produce the influenza virus for as long as 48 hours after infection. It was for the first time that the results denoted the identical susceptibility of primary pulmonary cells in mice and rats to influenza virus. A lower pulmonary susceptibility to influenza virus in rats versus mice could be indicative of that the surface factors of epithelial lining contribute essentially to shaping the pulmonary susceptibility to influenza virus since there is no difference of the susceptibility of pulmonary cells to influenza virus between the two above animals' species.

[Antiviral activity of oral ultralow doses of antibodies to gamma-interferon: experimental study of influenza infection in mice]. / Protivovirusnaia aktivnost' sverkhmalykh doz antitel k gamma-interferonu pri peroral'nom vvedenii: éksperimental'noe issledovanie grippoznoi infektsii u myshei.

Course intragastric administration of ultralow doses of human gamma-interferon antibodies (ULD anti-IFN-gamma) to intact mice resulted in an increase of endogenous IFN-gamma production by the animal lymphocytes. Oral prophylactic administration of ULD anti-IFN-gamma significantly lowered the influenza virus concentration in the animal lungs at the initial stage of the aerogenous infection: in 2 (p = 0.05) and 3 (p = 0.07) days after the contamination. The therapeutic antiviral effect of ULD anti-IFN-gamma in mice with influenza was evident from a significant decrease of the influenza virus concentration in the lungs of the animals on the 4th (p = 0.05) and 5th (p = 0.07) days after the contamination. The antiviral effect of ULD anti-IFN-gamma after the prophylactic and therapeutic use is likely provided by induction of endogenous IFN-gamma.

[Possibilities of culturing mutant variants of Ade12 adenoviruses in 293 cell culture]. / Vozmozhnost' kul'tivirovaniia mutantogo varianta Ade12 adenovirusa na kul'ture kletok 293.

The results of polymerase chain reaction and of DNA sequencing of the Adel2 mutant variant of adenovirus serotype 5, passaged 10 times and capable of selectively infecting and lysing the p53-deficient human tumor cells, are indicative of a high stability of its genotype and of the phenotypic properties acquired by it in successive passage on 293 cells. The absence of admixtures of wild-type adenovirus was clearly shown in the cultivation and passage processes. It was revealed in an experimental analysis of virus-productive properties of the studied continuous cell culture 293 by using the method of multilayer cultivation, that the maximal Adel2 yield is obtained at the 50% cytopathic effect. Virus doses, that are effective for cell-culture contamination, are within a range of 100-10 TCPE50 per cell. In order to spare the viral material, the infecting dose of 10 TCPE50 per cell was chosen to infect a cell monolayer.

[The simple method of direct estimation of infectious process in mice and rats aerogenically infected with influenza virus]. / Prostoi metod priamoi otsenki infektsionnogo protsessa u myshei i krys, aerogenno zarazhennykh virusom grippa.

Multiplication of influenza virus in laboratory animals (mice and rats) after aerogenic inoculation was recorded directly (by the agent accumulation in the lungs and trachea) and indirectly (by interferon concentration in the lungs of mice). Thermal inactivation of influenza virus in chick embryo allantoic fluid was observed (by 4.5-6 Ig within 48 h at 37 degrees C). The authors claim that influenza (strain A/Aichi/2/68) infection in the respiratory tract of mice and rats can be experimentally validated by inoculation of chick embryos with 10 and 20% mouse or rat lung homogenate (undiluted or diluted 10-fold) or with 1 and 5% mouse and rat trachea homogenate, respectively, 48 h after aerogenic inoculation of animals, and the virus AID50 be thus determined.

[Mechanisms of mice resistance to influenza virus A/AICHI/2/68 after preventive injection with polyprenols ]. / Mekhanizmy rezistentnosti myshei k virusu grippa A/AIChI/2/68 pri profilakticheskom vvedenii poliprenolov.

Preventive effect in influenza can be attained by intramuscular injections of fir (Abies) polyprenols. One of 5 tested polyprenol preparations (No. 1), injected 2 days before aerogenic infection with influenza virus, reliably protected mice from disease. Mice pretreated with polyprenol preparations or Hanks' solution did not differ by accumulation of interferon in the lungs One day after aerogenic infection. Three days after injection of polyprenol preparation No. 1 the weights of the spleen and thymus significantly decreased. One day after injection cell count in the bronchoalveolar tract of mice was almost 2-fold higher than in the control at the expense of lymphocytes and macrophages. After 3 days the relative and absolute counts of macrophages decreased and those of lymphocytes decreased significantly. Three days after injection macrophages were 2-fold more active in absorption of zymosan granules. Preparation No. 1 affected the production of superoxide anion radicals, whose production by all macrophages in the bronchoalveolar tract of mice was significantly higher on day 1 postinjection than on day 3 and higher than on days 1 and 3 after injection of preparation No. 2.