A new class of antivirals: antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells.

To enhance the penetration of oligonucleotide ('oligo') into cells, the oligo was combined with the hydrophobic undecyl residue. Using the 'DNA-synthesator', we synthesized oligo, complementary to the loop-forming site of the RNA, encoding polymerase 3 of the influenza virus (type A), and combined it with the undecyl residue added to the 5' terminal phosphate group. It was found that the modified oligo effectively suppresses the influenza A/PR8/34 (H1N1) virus reproduction and inhibits the synthesis of virus-specific proteins in MDCK cells. Under the same conditions, the non-modified antisense oligo and modified nonsense oligo did not affect the virus development.

Fatty acid acylated antibodies against virus suppress its reproduction in cells.

A method for suppression of virus reproduction in cells using fatty acylated antiviral antibodies, which in contrast to non-modified antibodies are capable of intracellular penetration, has been suggested. The addition of stearoylated antiviral antibodies to influenza A/Chili virus-infected cells causes a 100-fold suppression of virus reproduction. Non-modified antibodies do not produce any effect on virus reproduction.

Aprotinin aerosol treatment of influenza and paramyxovirus bronchopneumonia of mice.

The therapeutic efficacy of aerosolized aprotinin, a natural proteinase inhibitor, against influenza and paramyxovirus bronchopneumonia of mice is shown. Small-particle aerosol of aprotinin solution was generated by a Collison type nebulizer and infected mice were exposed to aerosol atmosphere by four 30-40 min incubations per day for 6 days. This regimen provided an inhalation aprotinin dosage of approx. 6 micrograms/mouse/day. With such treatment more than 50% of mice infected with lethal doses of either influenza virus or paramyxovirus were protected from death. A suppression of the development of fatal hemorrhagic bronchopneumonia and a normalization of the body weight gain were observed in infected mice treated with aerosolized aprotinin. These data suggest that low doses of aerosolized proteinase inhibitors could be successfully applied against respiratory influenza-like virus diseases.

Alphavirus replication in cultured cells and infected animals is inhibited by antiproteinase agents.

The influence of different antiproteinase agents on alphavirus replication was examined. Sindbis virus multicycle replication in cultured cells was suppressed by N-tosyl-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like proteinases, and by aprotinin, an inhibitor of a wide spectrum of proteinases. Antiviral activity of TPCK was also demonstrated in Sindbis virus-infected animals. Parenteral injections of TPCK in infected mice reduced virus titers in brain and blood. The possible mechanism(s) of antiviral action of the antiproteinase agents are discussed.

Hydrophobized antiviral antibodies and antisense oligonucleotides.

A method of suppressing virus reproduction in cells has been proposed. The approach consists of affecting the cells with antiviral antibodies artificially hydrophobized with fatty acid residues. Reproduction of influenza viruses in MDCK cells and respiratory-synticial virus in HeLa cells was used as a model to demonstrate that poly- and monoclonal antibodies, modified by 1 or 2 stearic acid residues, are potent, unlike the non-modified antibodies, at inhibiting viral reproduction. The observed phenomenon is apparently due to penetration of hydrophobized antibodies into the cells. Thus, in particular, considerable antiviral activity is exhibited by monoclonal antibodies against NP-protein of influenza virus, which is an antigen accessible to antibodies only inside the infected cells. Hydrophobized antibodies do not affect the kinetics of viral protein synthesis; they block the virus withdrawal from the cells, probably by interfering with the assembling and budding of virus particles. To enhance penetration of oligonucleotides ("oligos") into cells, chemical modification of the former at the 5'-end phosphate group by fatty radicals has been suggested. The undecanol-modified oligo namely an oligo complementary to the protein binding sites located at the influenza virus polymerases encoding RNA, was synthesized using a DNA-synthesator. The above modified oligo effectively suppressed the influenza A/PR8/34 virus reproduction and inhibited synthesis of the virus-specific proteins in MDCK cells. The non-modified antisense oligo and the modified nonsense oligo did not affect the virus development under the same conditions.

[Antiviral activity of proteinase inhibitors in cultured cells infected with alpha-viruses]. / Antivirusnoe deistvie ingibitorov proteinaz v kul'ture kletok, zarazhennykh al'favirusami.

The ability of synthetic inhibitors of trypsin-like (TLCK) and chymotrypsin-like (TPCK) proteinases and natural antiproteinase oligopeptides of animal (aprotinin) and microbial (enzistatin) origin to suppress multicycle replication of different alpha viruses (Semliki, Sindbis, Venezuelan equine encephalomyelitis viruses) in cultured cells was studied. Antiviral activity was found to be induced by TPCK and aprotinin (Gordox). These compounds were shown to reduce virus yield 100-fold and to prevent the involvement of cells into infection process. The mechanisms of antiviral activity and chemotherapeutic possibilities of antiproteinase compounds are discussed.

[Clinical effectiveness of aprotinin aerosol in influenza and parainfluenza]. / Klinicheskaia éffektivnost' aérozolia aprotinina pri grippe i paragrippe.

Aprotinin aerosol has been previously shown to have protective effects in experimental influenza- and parainfluenza-induced bronchopneumonias in animals. This paper presents the results of controlled clinical studies to evaluate the therapeutical efficiency of aprotinin aerosol in natural influenza and parainfluenza infections in human beings. A total of 52 patients were followed up. They received either soda (placebo) or aprotinin inhalations thrice a day for 4-5 days. The following mean duration (in days) of symptoms was found in the control (placebo-treated) and aprotinin-treated patients. These were: 2.5 versus 1.8 for fever, 2.0 versus 1.5 for headache, 2.9 versus 1.8 for weakness, 3.9 and 2.8 for common cold, 3.1 versus 1.6 for sore throat, 4.9 versus 2.8 for pharyngeal hyperemia, 4.9 versus 4.0 for cough, and 3.5 versus 1.3 for hoarse voice (p < 0.05). Inhaled aprotinin was well tolerated by the patients and caused no topical irritating effects and allergic reactions. The findings demonstrate the noticeable clinical efficacy of aprotinin aerosol in human influenza and parainfluenza.

[Cleavage of influenza virus hemagglutinin as affected by serum plasmin in cell culture and in vivo]. / Rasshcheplenie gemagglutinina virusa grippa pod deistviem syvorotochnogo plazmina v kul'ture kletok i in vivo.

The effect of chicken, canine, and porcine plasm containing plasmin proenzyme, plasminogen, on influenza virus hemagglutinin produced in homologous and heterologous tissue cells was studied. The cells incubated with the homologous plasm were found to produce virions containing both cleaved and uncleaved hemagglutinin whereas the cells incubated without plasm or with heterologous plasm produced virions with uncleaved hemagglutinin. The infectious activity of the virus produced by cells with the homologous plasm was much higher than that of the virus grown without the latter or with heterologous plasm. The addition to the culture medium of plasminogen inhibitors together with plasm eliminated the proteolytic effect of the plasm on virion hemagglutinins resulting in the production of virions with uncleaved hemagglutinin and low infectious activity. In vivo, in experimental influenza infection of mice and chickens, highly infectious virus with cleaved hemagglutinin was isolated from the organs of the animals. The organs of the animals inoculated with inhibitors of proteolytic enzymes yielded virus of low infectivity with uncleaved hemagglutinin. Administration of proteases inhibitors to the infected animals prevented the spread of virus infection in animals and had a therapeutic effect. The experimental data suggest that activation of virions with proteolytic enzymes of the host, in particular, plasmin by means of hemagglutinin cleavage is the key mechanism in the development and spread of influenza infection in the host.

[Inhibition of the reproduction of the influenza B virus by aprotinin]. / Ingibirovanie reproduktsii virusa grippa B aprotininom.

Injection of aprotinin, a natural inhibitor of proteinases, into the allantoic cavity of chick embryos infected with influenza B/Lee/40 or B/HK/73 virus resulted in inhibition of proteolytic cleavage of virus hemagglutinin HA into HA1 and HA2, thereby decreasing the level of proteolytic activation of the synthesized virus particles. As a result of this inhibition in aprotinin-treated embryos, multicycle virus reproduction was limited and virus yields decreased considerably. The experimental results indicate the potential of chemotherapeutic inhibition of infection caused by influenza B viruses using antiproteinase agents interfering with proteolytic activation of virions.

[The therapeutic effect of aprotinin inhalations in influenza and paramyxovirus infections in mice]. / Lechebnyi éffekt ingaliatsii aprotinina pri grippoznoi i paramiksovirusnoi infektsiiakh u myshei.

Mice infected with influenza A/Aichi/2/68 (H3N2) virus or Sendai/960 paramyxovirus were treated by inhalations of aerosol aprotinin, a broad spectrum inhibitor of proteinases. A course of inhalations of finely dispersed aerosol aprotinin including 4 exposures of 35-40 min each daily for 6 days provided respiratory administration of aprotinin in a dose about 100 kallikrein-inhibiting U/mouse per day. In mice treated by aprotinin inhalations, histological examinations showed decreased pulmonary pathology, and their body weights increased as much as in uninfected animals. In the placebo group, the weight decreased until the death of the animals. The protective effect of aprotinin inhalations was 40-80% with inoculation doses 10-100 MLD50/mouse. The treated animals died 2-4 days later than those in the placebo group. The results indicate the expedience of inhalation therapy with aerosol aprotinin in influenza and paramyxovirus respiratory infections.

[Suppression of the proteolytic activation of myxoviruses in infected chick embryos by using aprotinin]. / Podavlenie proteoliticheskoi aktivastsii miksovirusov v zarazhennykh kurinykh 'embrionakh s pomoshch'iu aprotinina.

The mandatory step in reproduction of myxoviruses (influenza viruses and paramyxoviruses) is proteolytic shearing of viral glycoproteins activating the infectivity of virions. Such activation of myxoviruses is realized by trypsin-like proteases of the host. This study demonstrated that proteolytic activation of virions could be inhibited by a physiological inhibitor of proteases, aprotinine. A single injection of aprotinine (preparations Gordox or Contrycal) into chick embryos infected with various influenza viruses (WSN/34, Udorn/72) and paramyxoviruses (Sendai/960, NDV/La Sota, NDV/Queensland) blocked shearing of viral glycoproteins, HA, FO, HNO as a result of which noninfectious virions with unsheared glycoproteins were predominantly synthesized. In aprotinine-treated embryos, multicycle virus infection was markedly decreased which led to the 10(4)-fold or greater reduction of the virus yield. The antiviral effect of protease inhibitors and possibilities of their practical use in viral diseases are discussed.

[Blocking of Sindbis virus replication in the body of infected mice using an inhibitor of chymotrypsin-like proteases]. / Blokirovanie replikatsii virusa Sindbis v organizme zarazhennykh myshei s pomoshch'iu ingibitora khimotripsinpodobnykh proteaz.

Parenteral administration of an inhibitor of chymotrypsin-like proteases (TPCK) to mice infected with alphavirus (Sindbis AR/339 strain) blocked virus replication in the brain and inhibited the development of viremia in the infected animals. The most likely mechanism of TPCK antiviral effect seems to consist in disturbance of proteolytic processing of viral proteins.

[Improved method of determining the infectivity of the influenza virus]. / Usovershenstvovannyi metod opredeleniia infektsionnoi aktivnosti virusa grippa.

The infectious activity of influenza A virus preparations with different ratios of unsplit (HA) and split (HA1 + HA2) hemagglutinin was studied. For this purpose the virus was cultivated in chick embryos (the virus with split hemagglutinin), chick fibroblast culture (unsplit hemagglutinin) and in chick fibroblast culture to the medium of which chick embryo allantoic fluid was added (partially split hemagglutinin). Proteins were analysed by polyacrylamide gel electrophoresis followed by the scanning of the gels. An improved plaque method in cell cultures under the agar overlay was used to assay the infectious activity of the virus preparations. This method gave more accurate determinations of the infectious titre of the preparations tested. The routine titration method gave higher infectious titres of the preparations particularly for the virus with unsplit hemagglutinin. Employing the new method, a ratio of infectious and physical particles in preparations with different HA/HA1 + HA2 contents was determined and the productive activity of cells of the chorioallantoic membrane in chick embryos and chick fibroblast cell cultures infected with influenza virus was evaluated.

[Antiviral and therapeutic action of protease inhibitors in viral infections: experimental and clinical observations]. / Protivovirusnoe i terapevticheskoe deistvie ingibitorov proteaz pri virusnykh infektsiiakh: éksperimental'nye i klinicheskie nabliudeniia.

The effect of protease inhibitors (gordox, contrycal, epsilon-aminocapronic acid) on the development of influenza virus-induced infectious process was studied. Administration of the above-mentioned drugs exerted a marked antiviral and therapeutic effect both in animal experiments and in treatment of children suffering from influenza. Electron microscopic examinations of lungs from influenza virus-infected animals treated with protease inhibitors revealed definite decrease in the number of virus particles and significant diminution of pathological lesions; the changes were noted which indicated activation of the body immune response. The use of a protease inhibitor, epsilon-aminocapronic acid, in children with influenza shortened the duration of influenza virus antigens persistence in the nasopharyngeal epithelium and reduced the duration of the disease symptoms by 1 1/2-2-fold. Administration of epsilon-aminocapronic acid in inhalations exerted most effective antiviral and therapeutic effect. The results obtained demonstrate the possibility of influenza treatment with inhibitors of proteolytic enzymes.

[The antiviral aerosol aprotinin. General effect on the body after inhalation administration]. / Antivirusnyi aérozol' aprotinina. Obshchee vozdeistvie na organizm pri ingaliatsionnom vvedenii.

A new medicine for the treatment of respiratory viral infections is described. It contains aprotinin, a natural inhibitor of proteases, and is used in the form of a fine aerosol for inhalation or intra-respiratory instillation. To estimate its innocuousness, the inhalation effect of the aerosol was studied on animals of two species. The experiments included examination of the functional state of the central nervous and cardiovascular systems, determination of the blood cell composition and biochemical blood count, morphological and histological investigation of the internal organs. The toxicological studies showed that aprotinin inhalation induced no adverse reactions which made it possible to recommend the aprotinin inhalation for the use in the treatment and prophylaxis of infections caused by a wide variety of respiratory viruses in humans.

[The pathogenetic therapy of acute respiratory diseases by aprotinin inhalations]. / Patogeneticheskaia terapiia ostrykh respiratornykh zabolevanii ingaliatsiiami aprotinina.

Inhalations of natural protease inhibitor aprotinin in the form of finely divided aerosol against acute respiratory infections caused by influenza virus, parainfluenza viruses, adenoviruses, and mixed infections produced subjective effect as early as the treatment day 1. Objectively, aprotinin therapy was associated with a 1.5-2-fold reduction in the duration of systemic and respiratory symptoms compared to placebo. As a rule, the inhalations were well tolerated and caused neither local irritation nor allergy. No hepatic, hematopoietic toxicity has been documented. Aprotinin inhalations are thought promising against influenza and acute respiratory infections.

[Aprotinin antiviral aerosol: study of the local irritating and allergenic action after inhalation]. / Antivirusnyi aérozol' aprotinina: izuchenie mestnorazdrazhaiushchego i allergiziruiushchego deistviia pri ingaliatsionnom vvedenii.

The aerosol of aprotinin, a natural low molecular weight polypeptide (m.w. about 160 kD) inhibiting a wide range of serine proteases may be used as an antiviral drug. The animal studies showed that it had no local irritating action on the mucosa. A long-term use of aprotinin in the form of a fine aerosol practically induced no allergenic side effects. The results of the study indicative of the absence of the allergic complications made it possible to recommend the aprotinin inhalations as a safe means in the treatment and prophylaxis of viral infections of the respiratory tract.

[The differential diagnosis of anthrax in man]. / K differentsial'noi diagnostike sibirskoi iazvy u cheloveka.

The paper reports group cases of the disease running as anthrax. The disease was not identified etiologically. The authors hold that it is important to make differential diagnosis of anthrax with necrobacillosis, pasteurellosis and contact pustular viral dermatitis. Laboratory and clinical diagnostic techniques are specified.

[The time characteristics of the exacerbation of chronic periodontitis and of odontogenic perimaxillary abscesses]. / Vremennye kharakteristiki obostreniia khronicheskogo periodontita i odontogennykh okolocheliustnykh abstsessov.

Time course of some laboratory values and clinical manifestations of inflammatory process is studied in 52 patients with exacerbations of chronic periodontitis and 284 patients with odontogenic perimaxillary abscesses. Routine laboratory studies (clinical and biochemical analyses of the blood and urine) were carried out. Acute odontogenic inflammations are characterized by an intermittent course with periodicity of about a week. The end of the first week from the disease onset coincided with recovery in exacerbation of chronic periodontitis and with the beginning of stabilization of inflammatory process in abscess. Recovery after abscess was observed 2 weeks after disease onset. These regularities may be useful for specifying the pathogenesis, terms of check-ups, and duration of treatment.