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The article provides a brief overview of the conceptual approach to the treatment of genital herpesvirus infections, a review of studies on the use of the interferon inducers Kagocel® in complex therapy of genital herpes, demonstrates clinical observations of a patient with recurrent genital herpesvirus infection, a patient with an exacerbation of genital herpesvirus infection that is difficult to respond to standard therapy, a patient with genital herpes, poorly controlled by acyclic nucleosides.

Activation of Toll-like receptors (TLRs) is one of the earliest indicators of functional activation of the innate immune system. Therefore, the development of drugs that stimulate the transcription of TLR/RLR genes and at the same time are “multi-target” drugs is an important task of modern immunopharmacology. In this regard, antiviral drugs that combine the properties of interferonogens and immunomodulators, which also include Cycloferon® and its analogues, are of great interest. The purpose of this study was to assess the expression of genes that determine the TLR/RLR signalling reactions of the innate immune system under the influence of immunomodulatory antiviral drugs based on acridoneacetic acid (Cycloferon® and Cycloferon L). The study was conducted using a model of immunocompetent cells: THP-1, differentiated by phorbol ester into macrophage-like cells. Gene expression analysis was performed using real-time polymerase chain reaction. The expression level of genes encoding TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and RIG-I was studied under the influence of the drugs Cycloferon® and Cycloferon L in three concentrations (156 μg/mL, 312 μg/mL and 625 μg/mL) on 1 hour, 4 hours and 24 hours. It was shown that the drug Cycloferon® at concentrations of 156, 312 and 625 μg/mL at 24 hours of exposure dose-dependently stimulated the expression of TLR2, TLR3, TLR4, TLR7, TLR8 receptor genes. A stable stimulation of the expression of RIG1 receptor genes was found upon exposure 4 hours to the drug in all studied concentrations. For the first time, it was revealed that the drug Cycloferon L stimulated a stable increase in the expression of TLR2, TLR3, TLR4, TLR7, TLR8 genes at an exposure period of 24 hours. Hereby, it was shown that the drugs Cycloferon® and Cycloferon L stimulated the expression of the TLR2, TLR3, TLR4, TLR7, TLR8 genes (and RIG1 for the drug Cycloferon), which are responsible for the synthesis of innate immune receptors.

The study of coronaviruses, including those capable of causing life-threatening diseases, continued for many decades. So did the study of interferons, as well as acridine acetic acid, which is a powerful interferon inducer. For a long time, both directions of research developed in parallel to each other. However, the discovery of SARS-CoV and the creation of Cycloferon based on acridine acetic acid made both research directions converge. To date, the abundance of factual and theoretical tenets is enough to estimate the potential effectiveness of acridine acetic acid against COVID-19.

The article presents the scientific substantiation of the use of interferon inducers in an infectious disease clinic the inducers of endogenous interferon of various chemical groups with antiviral action are examined and analyzed. The authors describe specific features of interferon induction by different drugs. They evaluate the effectiveness of cycloferon in the complex treatment of patients with influenza and other acute respiratory viral diseases, herpesvirus, arbovirus and intestinal infections, tuberculosis, including in the presence of HIV infection, chronic brucellosis, etc.

Aim. To assess the effectiveness of the use of the antiviral drug enisamium iodide in the complex treatment of acute respiratory viral infections (ARVI) caused by various pathogens in routine clinical practice. Materials and methods. А prospective randomized study included 134 patients who were treated in the epidemic season of influenza and ARVI in 20182019. All patients were examined for the presence of influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, rhinoviruses, adenoviruses in nasopharyngeal swabs by PCR. Patients of the main group received enisamium iodide along with symptomatic therapy, the control group received only symptomatic therapy. The primary parameter of the effectiveness of therapy was evaluated on the scale of the general severity of the manifestations of ARVI (Total Symptom Score TSS) from the 2nd to the 4th day and by the secondary criteria of effectiveness: assessment of the duration of ARVI, the severity of fever, the proportion of patients with normal body temperature, the duration of the main clinical symptoms of acute respiratory viral infections, the proportion of patients in whom complications requiring antibiotics were noted, the dynamics of interferon status on the 6th day. To conduct a statistical analysis, depending on the efficiency parameter, the ANCOVA method with a fixed group factor and an initial score on the TSS severity scale was used as covariates, a criterion for comparing quantitative indicators in two independent groups. Results. According to the results of the analysis of the primary efficacy parameter, the median (interquartile range) of the average score on the scale of the general severity of ARVI manifestations in the main group was 4.33 (3.675.83), in the comparison group 6.00 (4.677.25; p0.001). The duration of systemic and local manifestations of acute respiratory viral infections was statistically significantly less in the main group (p=0.002 and p=0.019, respectively). Prescription of additional therapy was required in 2 (2.9%) patients of the main group (patients taking enisamium iodide), compared with 8 (11.9%) patients in the control group. Serum levels of interferon  and interferon  on the last day of treatment were statistically significantly higher in patients of the main group compared with the control group (p0.001). Treatment (excellent) was evaluated by 42 (62.7%) patients, while in the control group only 17 (25.8%) patients gave similar ratings. Both patients (p0.001) and doctors (p0.002) rated therapy tolerance better in the study group. Conclusion. The results confirmed the safety and effectiveness of enisamium iodide as a treatment for ARVI and influenza. The antiviral, interferonogenic and anti-inflammatory properties of the drug are involved in the formation of an antiviral response and reduce the risk of complications, which makes it possible to reduce the number of symptomatic agents used.

The review is dedicated to the description of interferon inducers - new generation drugs created by Russian scientists as a result of the screening of thousands of high- and low-molecular synthetic and natural compounds. The drugs selected for clinical applications have a common ability to induce synthesis of endogenous interferons (IFN) after introduction into the organism. The main advantage of interferon inducers is a wide spectrum of antiviral activity, which is based on etiotropic and immunomodulatory effects of this group of drugs. Special attention is paid to the results of clinical application of interferon inducers.

The review contains a brief analysis of the 60-year history of the discovery, study and medical application of interferons, a new group of remarkable proteins that have found wide medical application in the therapy of virological, oncological, neurological, ophthalmic and other pathologies. Modern data on the classification of interferons and the mechanisms of their action are given. Particular attention is paid to the clinical use of medications of interferon and its inducers.

In the Russian Federation, the incidence of influenza and other acute viral respiratory infections (ARVI) increases every year during the autumn-winter period. The most common ARVI are parainfluenza, adenoviral and respiratory-syncytial infection. The constant mutability of influenza viruses and emergence of new ARI pathogens are especially dangerous. Medications from various groups including interferon inducers, particularly Kagocel, are recommended for the treatment and prevention of influenza and ARVI.

The article presents data on the place of the modern endogenous interferon inducers in the treatment and prevention of influenza and ARVI in children and the mechanisms of action. The focus is on the domestic antiviral innovation Kagocel which demonstrated high clinical efficacy and safety in a number of multi-center, blind, placebo-controlled studies of the treatment and prevention of ARVI/flu in children over 2 years old.

Acute infectious diarrheal diseases by the terminology of the World Health Organization or acute intestinal infections (AII) as they are called in Russia is a large group of acute infectious diseases characterized by development in patient of the diarrheal syndrome. The article provides statistical data on the incidence rate of AII among children that take one of the leading places in the structure of the infectious pathology and characterizing by the high mortality rate. Special attention is given to pathogenesis and therapy of acute viral intestinal diseases.

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The authors analyzed 254 cases of Enteroviral Meningitis (EVM) in children that were treated in the children's department of neuroinfections ofClinicalHospital№40 inClinical andDiagnosticCenter. We compared the conventional method of the treatment of EVM in children above three years with complex therapy that includes various immunotropic preparations. The efficiency of the therapy was estimated according to the results of clinical observations, and the parameters of systemic immunity. Basic therapy included the appointment of rehydration, vasoactive drugs, vitamins of group B. In comparison groups children were given one of the following preparations: Amixin, Anaferon for children, Cycloferon, Viferon in addition to the basic treatment. The study demonstrated the benefits of complex treatment for enteroviral meningitis with immunomodulators before applying basic therapy. Total maximum effect was observed in the group with Anaferon for children.

The article presents data on the place of the modern endogenous interferon inducers in the treatment and prevention of influenza and ARVI in children and the mechanisms of action. The focus is on the domestic antiviral agents Kagocel which demonstrated high clinical efficacy and safety in multi-center, blind, placebo-controlled studies involving treatment and prevention of ARVI/flu in children over 2 years old.

Abstract. Interferon profile was studied in 412 children and 41 adults with acute respiratory virus infections, as well as in forty-eight healthy children and ninety-nine adults. Appropriate interrelations were evaluated between interferon contents, and age, specific clinical features, etiology and clinical course of the disease. Therapeutic efficiency of interferons and interferon inducers was studied at 928 children. Some novel parameters are proposed to assess interferon state, i.e., stimulation index of interferon production, and interferon-producing activity of lymphocytes that represent individual functional activity of the patients’ immunocompetent cells. Criteria for interferon administration.

Aim: To assess the effectiveness of the use of the antiviral drug enisamium iodide in the complex treatment of acute respiratory viral infections (ARVI) caused by various pathogens in routine clinical practice., Materials and Methods: А prospective randomized study included 134 patients who were treated in the epidemic season of influenza and ARVI in 20182019. All patients were examined for the presence of influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, rhinoviruses, adenoviruses in nasopharyngeal swabs by PCR. Patients of the main group received enisamium iodide along with symptomatic therapy, the control group received only symptomatic therapy. The primary parameter of the effectiveness of therapy was evaluated on the scale of the general severity of the manifestations of ARVI (Total Symptom Score TSS) from the 2nd to the 4th day and by the secondary criteria of effectiveness: assessment of the duration of ARVI, the severity of fever, the proportion of patients with normal body temperature, the duration of the main clinical symptoms of acute respiratory viral infections, the proportion of patients in whom complications requiring antibiotics were noted, the dynamics of interferon status on the 6th day. To conduct a statistical analysis, depending on the efficiency parameter, the ANCOVA method with a fixed group factor and an initial score on the TSS severity scale was used as covariates, a criterion for comparing quantitative indicators in two independent groups., Results: According to the results of the analysis of the primary efficacy parameter, the median (interquartile range) of the average score on the scale of the general severity of ARVI manifestations in the main group was 4.33 (3.675.83), in the comparison group 6.00 (4.677.25; p0.001). The duration of systemic and local manifestations of acute respiratory viral infections was statistically significantly less in the main group (p=0.002 and p=0.019, respectively). Prescription of additional therapy was required in 2 (2.9%) patients of the main group (patients taking enisamium iodide), compared with 8 (11.9%) patients in the control group. Serum levels of interferon  and interferon  on the last day of treatment were statistically significantly higher in patients of the main group compared with the control group (p0.001). Treatment (excellent) was evaluated by 42 (62.7%) patients, while in the control group only 17 (25.8%) patients gave similar ratings. Both patients (p0.001) and doctors (p0.002) rated therapy tolerance better in the study group., Conclusion: The results confirmed the safety and effectiveness of enisamium iodide as a treatment for ARVI and influenza. The antiviral, interferonogenic and anti-inflammatory properties of the drug are involved in the formation of an antiviral response and reduce the risk of complications, which makes it possible to reduce the number of symptomatic agents used.

The review summarizes literature data on the development of drugs based on natural and synthetic high-polymeric double-stranded RNA, and their antiviral, immunoadjuvant and antitumor properties. Special attention is paid to cell receptors responding to exogenous dsRNA, the paths of dsRNA-dependent antiviral reaction, ability of dsRNA to inhibit growth and induce apoptosis ofmalignant cells. It has been shown that enhancing the innate immune response with dsRNA can be an effective component in improving methods for treating and preventing infectious and cancer diseases. The further use of dsRNA for the correction of pathological processes of different origin is discussed.

The paper deals with the influence of the adenovirus (Ad) and Epstein-Barr virus (EBV) on functional activity of lymphocytes, in particular, the production of alpha- and gamma-interferons, tumor necrosis factor (TNF) in conditions of mono- or double infection of B- and T-phenotype (CEM) lymphoblastoid cells. It is shown, that Ad, EBV or both viruses induce high enough levels of interferon on both lines of cells and in control epithelial cells. The lymphoblastoid cells infected by viruses deep ability to synthesize alpha- and gamma-interferons under the influence of the corresponding inducers (Newcastle disease virus and hemagglutinine). Nevertheless, the levels of their formation are not high. Rather high parameters of activity of the tumor necrosis factor (TNF) were revealed during a day in the initial B95-8 cells and superinfected Ad after the effect of LPS of E. coli. Their activity in CEM cells also did not depend on the infection type.

Interferonogenic acitivity of lacticacid bacteria was studied in experiments in vitro and in vivo. It have been shown, that interferon induction depends both on strain species and on culture medium used for lactobacteria harvesting. Streptococcus thermophilus induces interferon more actively in leucocytes (in vitro)--1280 AU/ml on the bines broth medium, and less actively--320 AU/ml on fermented corn medium. Interferon induction in human leucocytes was not registered in case of L. plantarum 200 strain. In vivo experiments show that E. faecium strain and strain mixture of streptococcus termophilus are active interferon inductors. Induced interferon is recognized as alpha- and gamma-interferon, with domination of alpha-interferon.

Evidence was obtained that the changed gravity, tension profiles of the magnetic fields inside the orbital station and other spaceflight factors (SFF) substantially influence the cell genome and synthesis of recombinant proteins. The authors proposed a technique of fixation of possible alterations in genes and expressed recombinant proteins in strains-producers of human alpha-interferon: HuIFN-alpha 2b, HuIFN-alpha 8a, HuIFN-alpha 10a, and HuIFN-alpha 14a. Spaceflight factors were simulated by way of gamma-irradiation at 10 Gy and 50 Gy by a cobalt unit, and centrifugation of samples at 10 g and 50 g. Cultivation of the strains-producers during the SFF simulation yielded HuIFN-alpha proteins with altered functional characteristics. Following exposure to simulated SFF, strains-producers expressed HuIFN-alpha that preserved a high titre of antiviral activity (AVA) but suppressed the antiproliferative activity (APA) inferring some structural/functional shifts in the HuIFN-alpha molecule due to, presumably, mutations in the active center responsible for APA. Determination of species specificity of the HuIFN-alpha recombinant proteins following exposure to SFF revealed dissociation of cross-AVA in homologous and heterologous cell cultures which can be also attributed to the structural/functional shifts in the HuIFN-alpha molecule. These changes can be localized in the receptor cluster of molecule or consequent to modification of the center defining HuIFN-alpha species specificity. The proposed simulation system allows fixation of shifts in the HuIFN-alpha structural/functional characteristics and investigations of the stability of eucaryotic genes in long-term space flights.

Influenza virus suppressed tumor growth after injection to tumor zone in a dose of 7-8 lg EID50, as was shown for two continuous mouse tumor cell strains, Ehrlich's carcinoma and L-1210 lymphoma. Influenza virus strains differed by their antitumor activity which correlated with their interferonogenic activity. Antitumor activity of influenza virus depended on the site of application, dose of the virus, and interferonogenic activity. Virus therapy of tumors stimulated specific cytotoxic activity towards tumor antigens.

Reviews the 40-year history of discovery and study of interferons. Traces the route from the first experimental observation to scientific interpretation of the phenomenon and its subsequent biological cognition. Pays special attention to modern stage in clinical use of interferons and their inductors and the contribution of Russian scientists to the problem.

Cells of monocytic-phagocytic series and immunocompetent cells derived from dogs: bone marrow, leukocytes, spleen, thymus, lymph nodes (with the exception of the liver) were found to be capable of producing interferon (IFn) in vitro in response to inoculation of viral and non-viral inducers. This capacity is manifested in different ways: thymus and lymph node cells were less active interferon producers. Studies of physicochemical properties of the resulting interferons confirmed the belonging of the virus-induced IFn to alpha-interferons, PHA-induced IFn to immune alpha-IFn. The above-mentioned refers both to bone marrow and leukocyte interferons.

Nonsteroid antiinflammatory agents (NAIA) such as antipyrine, butadion, acetylsalicylic acid, sodium salicylate, stampyrine and 4-iodantipyrine are not interferonogenic. Still, they stimulated interferonogenic action of poly(G).poly(C) in studies on animals. Relation between the interferon-stimulating action of the NAIA and their effect on activity of prostaglandin and the influence on the immune system was suggested.

The level and the time course of accumulation of endogenous interferon were studied in the blood sera of mice experimentally infected with Lassa virus by the intracerebral route. The level of interferon correlated with case-fatality rate of the animals which may be important for the understanding of the pathogenesis of arenavirus infections.

Double stranded RNA was isolated from bacteriophage phi 6 parasitizing on phytobacteria. Its interferon-inducing and antiviral activities were shown in vitro and in vivo. In the culture of L-929 cells, interferon resistant to heat and acids was synthesized. The interferon could be practically completely neutralized by specific anti-interferon serum. The phage phi 6 preparation dsRNA in the lyophilized form was studied. The preparation retained its biological activity. It was shown that a preparation containing 30 per cent of dsRNA was less toxic than a preparation containing 100 per cent of dsRNA, the difference in the interferon-inducing activity being insignificant.

The properties of lymphoid Namalwa cell line propagated at the USSR Academy of Medical Sciences Research Institute of Viral Preparations for interferon production are described. The scanning and transmissive electron microscopy studies of the cells showed their morphological stability and the absence of microbial contamination. The 46-48-chromosome cells comprised 85% of the population, hypodiploid cells (44-45 chromosomes), 9%, tetraploid and hypertetraploid cells, 3%. Spontaneous aberrations were detected in 3% of the chromosome. Inoculation of the cells into unsuppressed laboratory animals (rabbits, guinea pigs, adult or suckling mice) or chick embryos did not cause the development of any pathological process. Namalwa cells were shown to produce interferon after multiple (up to 4 times) induction with Newcastle disease virus.

The national preparation amixine induces interferon (IF) production in most of the people who have background values of serum IF. When the initial values of serum IF are high, after administration of amixine they decline to the background ones. A certain group of subjects (approximately 25%) is not sensitive to amixine. The IF content in the blood serum of such subjects does not change before or after administration of amixine. Its use should be limited to 4-5 administrations weekly, because its longer use leads to the exhaustion of the IF system in people.

The interferon-stimulating effect of immunomodulators is manifested after their combined inoculation with double-stranded NRAs (dsRNA) of natural and synthetic origin, and depends upon the time of administration of an inducer with respect to an immunomodulator and upon the dose of dsRNA. It is manifested as prolongation of interferon circulation in the bloodstream of animals up to 3 days when inosiplex is used as an immunomodulator. A still greater effect is observed when its derivative ASK is used instead of inosiplex. The interferon-stimulating effect of immunomodulators is due to cells of the immune system.

Comparative study of mitogenic and interferonogenic properties of staphylococcal enterotoxins of different serotypes is done. It is revealed that preparations of enterotoxins are polyclonal mitogens and have interferon-inducing activity. It is stated that enterotoxin of D type has the highest mitogenic activity, which is shown by interferon-inducing activity of A type toxin.

Antiviral activity of the complexes of synthetic polyribonucleotides, i.e. poly (I).poly (C) and poly (G).poly (C) obtained at non-equimolar ratios of homopolymers was studied. The system of chick embryon fibroblasts and horse Venezuellan eguine encephalitis virus served as the model. It was shown that the active and stable complexes poly (I).poly (C) and poly (G).poly (C) were formed at some excess of poly (C), i.e. at the ratio of poly G) or poly (I) to poly (C) equal to 40/60 to 20/80 molar per cent. The role of the excessive poly (C) in formation of the stable secondary structure of the nucleotide complexes and its significance as one of the means for affecting the fine structure of double-stranded RNA were discussed.

Spleen lymphocytes from normal subjects who suddenly died were used as cells producing gamma-interferon. One spleen yielded (3-8) X 10(8) viable cells which made it possible to prepare from 3 to 81 of splenocyte suspension culture. Staphylococcal enterotoxin A (SEA) and B (SEB) were used as gamma-interferon inducers. Stimulation of splenocyte suspension culture with SEA and SEB resulted in production of gamma-interferon with an average activity of 640-2560 units/ml. A partially purified interferon preparation with an activity of 2 X 10(4) units/ml was obtained by sorption of gamma-interferon on porous glass CPG-200-240 followed by elution with a buffer containing 50% ethylene glycol and Sephadex G-25 gel chromatography. As a result of 11 successive intramuscular immunizations of rabbits at 2-week intervals with a partially purified and concentrated preparation of gamma-interferon with Freund's complete adjuvant, blood serum was obtained which was capable of neutralizing 32 units of gamma-interferon up to a dilution of 1:128. The serum was highly specific: it showed no specific interaction with antigenic determinants of either natural alpha- and beta- or plasmid alpha-F and alpha-F/D human interferons.

The effect of a dsRNA preparation (an interferon inductor) on DTH induced by SRBC was studied. It was shown that at optimal antigenic load the dsRNA preparation inhibited DTH whereas at suboptimal and supraoptimal loads the preparation stimulated it. The findings indicated that the dsRNA preparation had an immunoregulatory effect. The immunoregulatory properties of the preparation must be associated with its action on the lymphocyte suppressor cells and macrophages.

Experiments in vitro were made to study adenosine deaminase (ADA) and 5'-nucleotidase (5-N) activity in NK after their treatment with interferon inductor--Newcastle disease virus (NDV) or with prostaglandin E2 (PGE2). It has been established that treatment of human NK with NDV leads to increasing of their cytotoxic activity (CTA), which is accompanied by rising of ADA activity and reducing of 5-N activity in these cells. Decrease of CTA under the influence of PGE2 occurs together with reduction of ADA activity and increase of 5-N activity in human NK. Changes in NK activity under the influence of various exogenous or endogenous factors may be due to the modification of activity of adenosine metabolism enzymes in these cells.

It is shown that prodigiosan is an inductor of synthesis of the substances with the thymosin-like activity. The suggestion about inhibition of 3-methylcholanthrene-induced blastomogenesis by prodigiosan which is mediated by the substances with the thymosin-like activity and interferon is discussed. We present the hypothetical diagram of the development of the above mentioned mechanism. The significance of the obtained findings for elaboration of the immunotherapy is discussed.

It was shown that levamisol administered orally to mice induced production of interferon with its maximum level in 4-6 hours and prolonged subsequent circulation in the host (the observation period of 5 days). Antiviral activity of levamisol in experimental forest-spring encephalitis was shown (protection of 35-40 per cent). When levamisol were used in combination with polyguacyl, an additive effect was recorded.

Antiviral activity of poly-I-poly-C complex with poly-L-lysine was studied on macaco rhesus. The complex bifilamentous polyribonucleotide induced active production of serum interferon and provided pronounced protection of the monkeys infected intracutaneously with the variolovaccine virus (10 LD50 for the monkeys in intracutaneous infection). The effectiveness of the protective effect depended on the scheme and route of the drug administration. The highest prophylactic and therapeutic effect was provided by local administration of the complex in a dose of I mg per I kg of the body weight, the incubation period being increased 2--3 times and the period of the skin affections being decreased approximately 2 times. The results of the studies on the effect of poly-I-poly-C complex with poly-L-lysine were evident of definite prophylactic activity of the drug against experimental vernal encephalitis in the monkeys. The animals not treated with the inductor died on the 16th or 17th day after infection because of the paralysis of the trunc and extremities muscles. The clinical evidences of the disease in the animals treated with the drug were not uniform: from complete health to death.

Daily administration of vaccine as well as combined use of antirabies gamma-globulin and vaccine were shown not to induce the production of immune T-killers possessing protective properties in adoptive transfer in mice pre-infected with street rabies virus in contrast to a single administration of a shock dose of antigen. Immune splenocytes protected 31-43% of mice infected with street rabies virus in a dose of 2-4 im LD50 and did not protect the animals infected with a dose of 12 im-LD50. After combined administration of immune splenocytes and an interferon inducer (double-stranded RNA) the additive protective effect was observed.

Conditions for and regularities of gamma-interferon production were elucidated. High interferon inducing activity of the known inductors such as concanavalin A, phytohemagglutinin and lentil lectin was asserted. It was shown that aqueous and alcoholic extracts of legumes seeds were also able to induce interferon synthesis in cultures of human peripheral blood leukocytes. By physicochemical properties such as stability to acids and liability to heating and by antigenic properties, interferon produced under such conditions was of type 2 (gamma). The maximum yield of interferon was observed in stationary and roller leukocyte cultures at a density of 2.10(6)-4.10(6) cells/ml on commercial rich media (IMDM and Eagle medium). Dynamics of gamma-interferon biosynthesis was determined. The peak of interferon production after the leukocyte induction with plant lectins was recorded in 72-96 hours.