Structural and evolutionary characteristics of HA, NA, NS and M genes of clinical influenza A/H3N2 viruses passaged in human and canine cells.

BACKGROUND: Canine (MDCK) cells and chicken eggs are usually used for isolation of human influenza viruses. Viruses isolated by these procedures often differ from those present in the clinical specimens, since adaptive changes occur during virus transmission from the human host to cells of heterologous origin. OBJECTIVES: To minimize these species-dependent changes, CACO-2 cells derived from human intestinal epithelium were used to isolate virus from influenza patients. STUDY DESIGN: Influenza A viruses of subtype H3N2 were primarily isolated in CACO-2 and then passaged in parallel in CACO-2 and MDCK cells. Structural properties of passaged virus variants were compared and analyzed for evolutionary relationships. RESULTS: Influenza viruses were isolated in CACO-2 with higher efficiency than in MDCK and chicken eggs. The following observations were made: (i) recent isolates showed an about 2-fold increase in the number of glycosylation sites of HA and NA when compared to isolates from 1968 to 1970; (ii) during passages of clinical strains in CACO-2 and MDCK cells HA and NA mutated cooperatively with strain-specific variations implying that functioning of the HA-NA complex varied from strain to strain in one influenza outbreak; (iii) there were no amino acid exchanges in the HA receptor binding site although the viruses acquired the ability to agglutinate avian erythrocytes after passage in MDCK cells, suggesting that virus adsorption is regulated by several factors; (iv) quasispecies characterized by deletion of 66 nucleotides (22 amino acids) in the stalk region of the NA gene was dominant in naso-pharyngeal washes of all patients whereas during passaging in CACO-2 cells this deleted genotype in isolates from different patients was either stably retained as prevalent quasispecies or rapidly replaced for that one containing full length NA gene; (v) the M2 protein of clinical viruses was sensitive to amantadine; (vi) the NS segment of human viruses, unlike the most of avian ones, contained an additional positive-sense open reading frame encoding a hypothetical 25kD polypeptide (negative strand protein, NSP). CONCLUSIONS: The data suggest that (i) clinical influenza viruses can be isolated from respiratory tract of humans more effectively in human than in canine cells; (ii) heterologous virus population circulates during one influenza outbreak; (iii) increasing numbers of glycosylation sites on HA and NA and stalk shortening of NA take place during virus evolution in humans.

Specific biochemical features of replication of clinical influenza viruses in human intestinal cell culture.

Influenza A viruses isolated from the respiratory tract of patients with influenza were cultured in human intestinal epithelium cells (CACO-2 line). The CACO-2 cells were found to be 100-fold more susceptible to the clinical viruses than MDCK cells and chicken embryos. On passaging in CACO-2 cells, clinical isolates of the subtype H3N2 retained the original "human" phenotype and agglutinated human but not chicken erythrocytes, whereas on passaging in MDCK cells the virus phenotype changed to the "avian" one. On comparison with laboratory strains (grown in chicken embryos or MDCK cells), the clinical viruses were characterized by higher stability of the anti-interferon protein NS1 but had a reduced synthesis of the matrix protein M1, and this could facilitate the virus adaptation and escape of the infected cells from immune attack in the human body. The increased tropism to the human CACO-2 cells correlated with higher adsorption of the clinical viruses on cellular receptors. However, in the CACO-2 and MDCK cells the ratio of sialyl-containing glycoreceptors of the 2-3 and 2-6 type was similar. These observations indicated that not only sialic acid residues were involved in the adsorption and penetration of the clinical viruses into human cells, but also the protein moiety of the cellular receptor itself and/or an additional cellular coreceptor. Thus, clinical influenza viruses are shown to possess a specific mechanism of sorption and entry into human epithelial cells, which is responsible for their higher tropism to human cells and is unlike such a mechanism in canine cells.

Intracellular cleavage of human influenza a virus hemagglutinin and its inhibition.

Replication of human influenza A viruses and proteolytic cleavage of the viral glycoprotein HA0 -->HA1/2 were studied in passaged cultures of epithelial cells of the mucosal membrane of human large intestine (CACO-2 line), dog kidney cells (MDCK), and monkey kidney cells (CV-1). Cleavage of the viral glycoprotein HA0, synthesis of activated virions, multicycle virus infection, and effective production of viral foci under an agarose overlayer were found in CACO-2 cells. By pulse-chase labeling of viral glycoproteins, testing the sensitivity to endoglycosidase-H of the viral glycoproteins HA0 and HA1/2 synthesized, and inhibiting the HA0 proteolysis with brefeldin A, the HA0 -->HA1/2 proteolysis was established to occur in the late stages of intracellular transport in the trans-Golgi and plasma membrane areas of the cells. Proteolysis of the viral glycoprotein HA0 in CACO-2 cells was suppressed by aprotinin, a natural inhibitor of serine proteinases. Unlike MDCK and CV-1 cells resistant to apoptosis induced by influenza virus, CACO-2 cells retained their viability for 2-3 days after infection with human influenza A virus.

[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.

[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.

[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 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.

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.

Replication of influenza B virus in chicken embryos is suppressed by exogenous aprotinin.

Chicken embryo proteinases, one of which is a blood clotting factor Xa-like proteinase, are known to effectively cleave the haemagglutinin (HA) of Influenza B viruses to permit their replication in chicken embryonated eggs. Here we show that injection of the serine proteinase inhibitor, aprotinin, into the allantoic cavity of eggs infected with Influenza B/Hong Kong/73 and B/Lee/40 viruses suppresses the viral HA cleavage and reduces the virus proteolytic activation and replication. Effective inhibition dose was determined as approximately 10.0 micrograms of aprotinin per embryo that corresponds to 0.1 microM concentration. However, heparin, which is known to be a direct inhibitor of the Factor Xa, was not able to suppress Influenza B virus hemagglutinin cleavage and replication in chicken embryo system. These data shed light on the pattern of proteinases involved in the Influenza B virus proteolytic activation and indicate that aprotinin possesses antiviral potential against Influenza B viruses.

[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.

[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.

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.

[Cytologic diagnosis of thyroid gland adenoma and goiter]. / Tsitologicheskaia diagnostika adenomy i zoba shchitovidnoi zhelezy.

Examinations of smear impressions of postoperative biopsy specimens of the thyroid from adenoma patients have revealed among polygonal structures 1 to 20 spherical complexes (per micropreparation) consisting of relatively monomorphic follicular cells with intensively blue cytoplasm. The 'ball' size was 200-900 micron or more. Cellular composition, specific features of cellular structure, and the presence of spherical structures permit differentiating adenoma from goiter.

Effective inhibition of viral reproduction by hydrophobised antiviral antibodies.

A method is proposed for the inhibition of viral reproduction in cells by means of fatty-acylated antiviral antibodies which, in contrast to the unmodified antibodies, have the ability to enter the cells. The potential of this technique is demonstrated in experiments involving inhibition of the reproduction of various strains of influenza virus and respiratory syncytial virus.

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.