A new mouse unilateral model of diffuse alveolar damage of the lung.

OBJECTIVE AND DESIGN: The existing biological models of diffuse alveolar damage (DAD) in mice have many shortcomings. To offset these shortcomings, we have proposed a simple, nonsurgical, and reproducible method of unilateral total damage of the left lung in ICR mice. This model is based on the intrabronchial administration of a mixture of bacterial lipopolysaccharide (LPS) from the cell wall of S. enterica and α-galactosylceramide (inducing substances) to the left lung. METHODS: Using computer tomography of the lungs with endobronchial administration of contrast material, we have been able to perform an operative intravital verification of the targeted delivery of the inducer. The model presented is characterized by more serious and homogeneous damage of the affected lung compared to the existing models of focal pneumonia; at the same time, our model is characterized by longer animal survival since the right lung remains intact. RESULTS: The model is also characterized by diffuse alveolar damage of the left lung, animal survival of 100%, abrupt increases in plasma levels of TNFa, INFg, and IL-6, and significant myocardial overload in the right heart. It can be used to assess the efficacy of innovative drugs for the treatment of DAD and ARDS as the clinical manifestations that are developed in patients infected with SARS-CoV-2. Morphological patterns of lungs in the noninfectious ("sterile") model of DAD induced by LPS simultaneously with α-galactosylceramide (presented here) and in the infectious model of DAD induced by SARS-CoV-2 have been compared. CONCLUSION: The DAD model we have proposed can be widely used for studying the efficacy of candidate molecules for the treatment of infectious respiratory diseases, such as viral pneumonias of different etiology, including SARS-CoV-2.

[Virus specific antibody - based remedies for the urgent prevention and treatment of Ebola virus disease].

The Ebola virus (member of Ebolavirus genus Filoviridae family) is the etiologic agent of extremely hazard human disease with high mortality rates (up to 90%). The most important components of spectrum of therapeutics for special prophylactic and current of disease, caused by Ebola virus, are prepares, based on virus specific antibodies (convalescent's plasma, geterologic immunoglobulins, monoclonal antibodies. The use of different class therapeutics, based on virus specific antibodies, the possible improvements of its composition and strategy of its application for special prophylactic and current of disease, caused by Ebola virus, are considered in this review.

[The use of genetic engineering constructions as control samples on evaluation of diagnostic kits for reveal of rna of hazard and extremely hazard agents of virus infections by reverse transcription - polymerase chain reaction.]

The reverse transcription - polymerase chain reaction method (RT-PCR) has leading position on diagnostic infections, caused by RNA-containing viruses. This method presents severe requirements to carrying out of everybody stages of analysis (extraction of nucleic acid, carry out reverse transcription, amplification of DNA). It is necessary to account the possibility of false positive or false negative results appearance. The use on RT-PCR only positive (PCS) and negative (NCS) control samples is insufficient for the control of stages of RNA extraction and reverse transcription. That is way there is necessity the construction of inner control sample (ICS) to control of these stages. The main goal of present is the ground of use genetic engineering constructions (GEC) as control samples (PCS and ICS) on evaluation of diagnostic kits for reveal of RNA of hazard and extremely hazard agents of virus infections by RT-PCR. The vector recombinant plasmids, containing the insertion of cDNA of agent´s genomic RNA are used as PCS, RNA was packed in membrane protein of MS2 bacteriophage, is used as ICS. It is demonstrated that ICS does no influence on sensitivity of RT-PCR both for use of native agents and for use of synthetic nucleic acids of Ebola, Marburg, Lassa, Machupo, Venezuelan encephalitis equine (VEE), Rift Valley fever and rabies viruses. The possibility of use of PCS and ICS for standardization of diagnostic kits is discussed.

[Lujo hemorrhagic fever].

Lujo hemorrhagic fever (LHF) is a viral disease accompanied with fever, headache, vomiting, diarrhea, arthralgia, myalgia and numerous signs of hemorrhagic syndrome. LHF causes a clinical syndrome remarkably similar to Lassa hemorrhagic fever. The first case of LHF occurred in Johannesburg, South Africa, in 2008. There was a secondary transmission from the index patient to four healthcare workers. Four of the five patients died. The etiologic agent of LHF is Lujo virus (LUJV) belonging to Arenavirus genus of the Arenaviridae Family. Virus Lujo is the second pathogenic arenavirus, after Lassa virus, to be recognized in Africa during the last 40 years. Data about epidemiology, clinical characteristics and diagnostics of LHF, properties of Lujo virus (according to phylogenetic analysis), and recommended precautions for preventing secondary transmission are considered in this paper.

An experience in the clinical use of specific immunoglobulin from horse blood serum for prophylaxis of Ebola haemorrhagic fever.

The aim of this work was to estimate the efficacy and safety of single intramuscular introduction of specific heterologous immunoglobulin as prophylactic drug against Ebola hemorrhagic fever. Materials and methods. The specific heterologous immunoglobulin was introduced as a special prophylactic drug to 28 patients in epidemic situations, after skin hurt with infectious materials or contact with infectious blood. Clinico-laboratory observation was performed in 24 subjects after single intramuscular introduction of heterologous immunoglobulin Ebola. The samples of blood serum were investigated for immunoglobulin Ebola and antibodies to horse gamma-globulin on the 30th and 60th days after prophylaxis. Results. None of the subjects of the study contracted Ebola fever. There were no anaphylactic reactions after special prophylaxis with specific heterologous immunoglobulin. Among the subjects with normal allergic state 31% responded with local reactions; 13%, with a general reaction (mild case of the serum disease). Almost no reaction was observed in patients with unfavorable allergic state subjected to desensitizing therapy; in the absence of desensitizing therapy, 50% of patients with unfavorable allergic state exhibited local reactions; 17%, mild cases of the serum disease; 33%, moderate cases of the serum disease. In summary, if the tactics of immunoglobulin application was right, the quantity of local allergic reactions was 28%; of wide spread reactions, 6%. Weak serum disease was observed in 11% of the subjects. The prognostic period of resistance to Ebola fever was less than 30 days. Conclusion. The prophylactic use of specific immunoglobulin from horse blood serum against hemorrhagic Ebola fever is effective and relatively safe in patients subjected to desensitizing therapy.

[THERMAL STABILITY AS A PROGNOSTIC INDICATOR OF CONSERVATION OF LIVE EMBRYONIC SMALLPOX VACCINE (TEOVAC) DURING STORAGE].

AIM: Determination of values of coefficients of thermal stability of TEOVac for prognosis of conservation of the vaccine (specific biological activity) during the process of warranty period storage. MATERIALS AND METHOD: TEOVac (masticatory tablets) in primary packaging was kept at increased temperature (accelerated and stress-tests) and at the conditions established by PAP for the preparation (long-term tests). Biological activity of the vaccine was determined by titration on 12-day chicken embryos. RESULTS: A correlation between the value of coefficients of thermal stability and conservation of the prepared series of the condition preparation at the final date of storage was experimentally established. CONCLUSION: Coefficients of thermal stability could be used as a prognostic indicator of quality of the produced pelleted formulation of the preparation for evaluation of conservation of the vaccine during warranty period storage.

[The vaccines based on the replicon of the venezuelan equine encephalomyelitis virus against viral hemorrhagic fevers].

The status of the various recombinant DNA and RNA-derived candidate vaccines, as well as the Venezuelan equine encephalomyelitis virus (VEEV) replicon vaccine system against extremely hazardous viral hemorrhagic fevers, were reviewed. The VEEV-based replication-incompetent vectors offer attractive features in terms of safety, high expression levels of the heterologous viral antigen, tropism to dendritic cells, robust immune responses, protection efficacy, low potential for pre-existing anti-vector immunity and possibility of engineering multivalent vaccines were tested. These features of the VEEV replicon system hold much promise for the development of new generation vaccine candidates against viral hemorrhagic fevers.

[Sensitivity and specificity of the ELISA Kit for the detection of antidobies to Junin virus].

The goal of this work was to describe methodological approaches to determination of sensitivity and specificity of the enzyme-linked immunosorbent assay kit (ELISA Kit) for detection of the specific anti-Junin virus (JV) antibody. Comparison of ELISA to plaque reduction neutralization test (PRNT) showed direct relationship between antibody titers in the samples of serum of immunized animals, determined by either PRNT or ELISA methods. The obtained results provided an opportunity to form the panels of positive and negative serum samples to determine the sensitivity and specificity of the ELISA Kit. Sensitivity of the ELISA Kit was at least 98% when studying the samples of serum of immunized guinea pigs and rabbits (determined as positive in PRNT). The sensitivity of the ELISA Kit was at least 68% when studying the samples determined by PNRT as uncertain positive. The specificity was 98%. The specificity of the ELISA Kit was 98%.

[EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION].

AIM: Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. RESULTS: An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. CONCLUSION: During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.

[EPIDEMIOLOGIC ANALYSIS OF OUTBREAKS OF DISEASES CAUSED BY AMERICAN EQUINE ENCEPHALITIS CAUSATIVE AGENTS IN ENDEMIC REGIONS].

Epidemiologic analysis of epidemic outbreaks caused by American equine encephalitis causative agents is carried out in the review. Eastern equine encephalomyelitis (EEE), Western equine encephalomyelitis (WEE) and Venezuela equine encephalomyelitis (VEE) viruses are etiologic agents of dangerous transmissive diseases that are usually accompanied by fever and neurologic symptoms. Among the New World alphaviruses, VEE virus has the most potential danger for humans and domestic animals. Currently, enzootic strains of VEE play an increasing role as etiologic agents of human diseases. Most of the VEE cases in humans in endemic regions during inter-epidemic period are caused by infection with VEE subtype ID virus. A possibility of emergence of novel epidemic outbreaks of VEE is determined by mutations of ID subtype strains into IC subtype, and those currently pose a potential threat as an etiologic agent of the disease. Despite low morbidity, EEE and WEE are a problem for healthcare due to a relatively high frequency of lethal outcomes of the disease.

[DETECTION OF VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS RNA IN BIOLOGICAL SAMPLES BY REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION].

AIM: Detection-and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). MATERIALS AND METHODS: VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating. and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. RESULTS: VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. CONCLUSION: Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.

[Methods and Means for Defense from Collateral Action of Vaccine Virus after Vaccination Against Spallpox].

In connection with actuality of smallpox vaccination at present its methods and means are discussed to increase the safety of the first immunization with live vaccines.

[Investigation of Therapeutic Efficacy of Triazavirin Against Experimental Forest-Spring Encephalitis on Albino Mice].

The comparative study of the therapeutic efficacy of Triazavirin against experimental Forest-Spring encephalitis on albino mice vs. the active drug Ribavirin® showed that in high doses (200-400 mg/kg) Triazavirin moderately protected the infected animals. A significant increase of the animal lifespan in the test groups (from 4.1 to 4.8 days) and a statistically (p ≤ 0.05) valid decrease of the virus accumulation in the target organ (the brain) were observed.

[Investigation of Prophylactic Efficacy of Triazavirin Against Experimental Forest- Spring Encephalitis on Albino Mice].

Prophylactic efficacy of Triazavirin against experimental Forest-Spring encephalitis was studied on albino mice. vs. the active drug Ribavirin. A significant increase of the animal lifespan in the test groups (from 4 to 5 days) and a statistically (p < or = 0.05) valid decrease of the virus accumulation level in the target organ (the brain) were observed.

[Vaccine-like viruses: peculiarities of circulation in the South America].

The peculiarities of the spread of vaccine-like viruses first revealed more than 50 years ago in the area of the South America was discussed. These viruses cause infective episodes among milk cattle and caretaking personnel. Cancellation of the smallpox vaccination in 1980 resulted in a decrease in the community immunity and increased the risks of human infection. This circumstance makes it necessary to activate monitoring of the properties of the vaccine-like viruses, the circle of hosts and possible changes in the pathogenicity for humans.

[Study of sensitivity of laboratory animals to a causative agent of argentine hemorrhagic fever].

AIM: Study sensitivity of laboratory animals to a causative agent ofArgentine hemorrhagic fever. MATERIALS AND METHODS: Junin virus strain XJ P37 was obtained from the State Collection of Causative Agents of Viral Hemorrhagic Fevers of the Pathogenicity Group I of Scientific Research Center of the 33rd Central Scientific Research Test Institute (SRC of the 33rd CSRTI). Junin virus strain XJ P37 culture with biological activity of 5.2 1g PFU x ml was used in the experiments. Mice (2 - 4 and 7 - 14 days old), guinea pigs (250 - 300 g), 1.8 - 2.5 kg shinshilla breed rabbits, 2.0 - 3.0 kg javanese macaque monkeys were obtained from vivarium of the SRC of the 33rd CSRTI. Vero (B) and GMK-AH-1 (D) cell cultures were obtained from cell culture collection of the SRC of the 33rd CSRTI. Biological activity calculation of Junin virus was carried out by Kerber in I.P. Amsharin modification. RESULTS: Lethality in animals was from 12.5 to 50% after intranasal and intraperitoneal infection of guinea pigs, intramuscular, intraperitoneal and subcutaneous infection of rabbits, intracerebral and intranasal infection of mice at the doses from 0.4 to 1.0 x 10(5) PFU. Death of infected monkeys after intramuscular administration of the virus at 1.0 x 10(4) PFU dose was not observed. In 100% of surviving animals formation of virus-neutralizing antibodies was registered. CONCLUSION: Evaluation of sensitivity of laboratory animals to Junin virus has shown that intracerebrally infected mice may be used to maintain causative agent culture, infected guinea pigs - to prepare virus-containing cultures and modelling infection exacerbation in humans. Intramuscularly infected rabbits may be used to obtain hyper-immune sera.

[Experimental estimation of immunomodulating activity of Tubosan, a new Russian synthetic drug].

Tubosan, a new Russian immunostimulator is a derivative of sulfopyrimidine. It stimulated the humoral antiviral immunity in mice, provoked increasing the number of the antibody forming cells in the spleen of mice, immunized by sheep's erythrocytes, and increased the titers of the virus-neutralizing antibodies after immunization by inactivated and live vaccines VEE. The Tubosan ability to lower the reactogenicity of the live VEE vaccine provided new perspectives for using such immunomodulators in medical practice.

[Investigation of Triazavirin antiviral activity against tick-borne encephalitis pathogen in cell culture].

The efficacy of Triazavirin against the tick-borne encephalitis virus was estimated in the sensitive cell culture vs. the active drug Ribavirin. In a concentration of 128 mcg/ml Triazavirin was shown active in inhibition of the tick-borne encephalitis virus reproduction (strain Sofiin) by accumulation in the SKEV cell culture.

[The molecular genetic characteristic of RNA of human enterovirus detected in bio-test from child with serous meningitis].

The article considers molecular genetic characteristic of RNA of human enterovirus detected in bio-test from child with serous meningitis. The nucleotide sequence of genome DNA is analyzed. In 98% it is identical to corresponding nucleotide sequences of strains of human enterovirus A serotype 71 detected in China.

[The separation, identification and molecular biologic analysis of isolates of pandemic influenza virus A(H1N1)pdm09].

The article presents data concerning analysis of bioassays under suspicion of influenza A(H1N1)pdm09. The technique of back transcription polymerase chain reaction and sequencing was applied. The separation of native influenza virus A was implemented using passaging through developing chick embryos.