The influenza A virus NS genome segment displays lineage-specific patterns in predicted RNA secondary structure.

BACKGROUND: Influenza A virus (IAV) is a segmented negative-sense RNA virus that causes seasonal epidemics and periodic pandemics in humans. Two regions (nucleotide positions 82-148 and 497-564) in the positive-sense RNA of the NS segment fold into a multi-branch loop or hairpin structures. RESULTS: We studied 25,384 NS segment positive-sense RNA unique sequences of human and non-human IAVs in order to predict secondary RNA structures of the 82-148 and 497-564 regions using RNAfold software, and determined their host- and lineage-specific distributions. Hairpins prevailed in avian and avian-origin human IAVs, including H1N1pdm1918 and H5N1. In human and swine IAV hairpins distribution varied between evolutionary lineages. CONCLUSIONS: These results suggest a possible functional role for these RNA secondary structures and the need for experimental evaluation of these structures in the influenza life cycle.

[ANTIVIRAL ACTIVITY OF THE DIHYDROQUERCETIN DURING THE COXSACKIEVIRUS B4 REPLICATION IN VITRO].

A study of the antiviral activity of antioxidants against viral infections is believed to be essential for creating complex antiviral agents. Dihydroquercetin is considered as the most active antioxidant extracted from Larix gmelinii. In this work, we present results of experiments of the antiviral properties of dihydroquercetin against a member of the family Picarnaviridae--Coxsackievirus B4 in vitro. We have estimated that dihydroquercetin reduces viral titers at 100 µg/ml concentration as compared with control of virus. We have shown using the plaque assay that CPE of virusis reduced in the presence of dihydroquercetin at 100 µg/ml. Study of the phase of viral lifecycle, in which dihydroquercetin acted, demonstrated that the highest efficacy of the antiviral therapy was reached at early stages of virus reproduction (1-3 hours post infection). These results show that dihydroquercetin has antiviralproperty against Coxsackievirus B4. This drug and other antioxidants can be tested as inhibitors of viral replication.

[Adenosine A2A receptor as a drug target for treatment of sepsis].

Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A(2A) receptor (A(2A)AR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A(2A) receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A(2A)AR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A(2A)AR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis.

CHRONIC HEPATITIS WITH DOUBLE B/C INFECTION: VIROLOGICAL, CLINICAL, MORPHOLOGICAL CHARACTERISTICS.

AIM: To estimate the r, virological and clinical characteristics of chronic viral hepatitis (CVH) with double B/C infection. MATERIALS AND METHODS: We examined 282 patients with CVH. Genomes of hepatitis B virus (HBV) and hepatitis C virus (HCV) were studied by PCR in blood and liver (AmpliSens HBV and Amplisens HCV Russia), nuclear proteins (HBcorAg HBV and NS3 HCV) were determined by immunohistochemical method (Novocastra, UK), HBVgenome was sequenced by the Sanger method using ABI prism BigDye Terminator v3.1 kits and ABIPRISM 3100 analyzer (AppliedBiosystems, USA). Indices of histological activity (HAI), fibrosis, and portal vein (PV) congestion index (CI) were calculated by formula CI=SBB/LB V where S is P V cross section area in cm2 and LB V - linear blood flow velocity in cm/s (Vivid Pro- 7 apparatus, USA). RESULTS: CVH with double B/C infection was diagnosed in 85 (30.1%) patients including 44.7% with viral genomes and proteins in the live; 42.4% with HCVviremia, and 12.9% with HBJV/HCVviremia. Maximum CVH activity was documented in patients with latent HBV/HCVviremia (ALT 157.2±59.2 U/, HAI 11.6±1.3,fibrosis 2.8±0.7, C1 0.059±0.005); it was minimal inpatients.without viremia (Alt 76.25±63.0 U/I, HAI 6.7+-0.6,fibrosis 1.7±0.5, CI 0.042±0.001;p <0.05). Patients with latent HBV infection had precore/ore and pres/s mutations in HBVgenome and cytoplasmic localization ofHBcorAg. CONCLUSION: Double B/C infection was diagnosed in 30.1% of the patients with CVH dominated by HCV Patients with latent HBVhadprecore/ore and pres/s mutations. The highest intensity of hepatic cellular inflamation,fibrosis, and PV congestion was associated with HBV/HCV viremia and the lowest with intrahepatic localization of both viruses.

[Oncolytic potential of recombinant influenza A virus vectors on a model of malignant glioma in vivo].

Malignant glioma is the most frequently occurring primary brain tumor. Despite significant progress in the diagnostics and treatment of neoplastic diseases the prognosis for patients with III-IV grade gliomas, remains extremely unfavorable. Rapidly developing area in oncology is the employment of therapeutic viruses with natural or genetically engineered oncolytic activity. In the present study we demonstrated the oncolytic potential of a recombinant influenza A virus vector with impaired interferon antagonism function of NS1 protein in treatment of malignant glioma. Recombinant influenza A virus (HA-DS-GFP) expressing green fluorescent protein from the NS1 open reading frame was used as a model vector. HA-DS-GFP virus has shown infectivity towards glioma cells both in vitro, and in vivo (experimental glioma model in rats). Intratumoral inoculation of HA-DS-GFP resulted in a substantial inhibition or complete regression of tumor growth. Our data demonstrate that recombinant influenza vectors have promising potential in therapy of malignant gliomas.

[Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents].

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

[Comparison of the influenza epidemics in Russia caused by the pandemic virus A(H1N1)pdm09 within the period from 2009 to 2013].

Comparative analysis of the three past epidemics with the participation of the pandemic influenza A(H1N1)pdm09 was conducted according to the results of the epidemiological trials of two WHO National influenza centers for the morbidity, hospitalization, and mortality of the influenza in 59 cities of Russia for the period from 2009 to 2013. The first wave of the pandemic of 2009 was the most severe. Compared with this wave, during the next epidemics of 2011 and 2013, the involvement of urban population in the epidemic was reduced, as well as the morbidity in the people 15-64 years old and schoolchildren 7-14 years old. The duration of the epidemic among the adult population, the mortality rate of the total population, and the mortality rates in all age groups were also decreased. Vice versa, the incidence in the children of preschool age and the elderly people and the duration of the epidemic among children (especially preschool children) were increased. The share of patients 65 years and older, children 0-2 years old, and patients with pathology of the cardiovascular systems among the deceased patients increased to 33.6%.

[Clinical efficacy of arbidol (umifenovir) in the therapy of influenza in adults: preliminary results of the multicenter double-blind randomized placebo-controlled study ARBITR].

AIM: To evaluate the efficacy and safety of Arbidol (umifenovir) in adult patients with influenza. SUBJECTS AND METHODS: The analysis of the preliminary results of the multicenter double-blind randomized placebo-controlled post-marketing study ARBITR was performed. A total of 293 adults aged 18 to 65 years with influenza or acute respiratory tract infection of no more than 36 hours' duration were enrolled in the study. Individuals were randomized into 2 treatment groups: oral umifenovir 200 mg four times daily for 5 days or placebo four times daily for 5 days. The efficacy endpoints were time to resolution of all symptoms, severity of symptoms and illness, durations of virus shedding. RESULTS: The efficacy of umifenovir was evaluated in the group of 119 (40.6%) patients with influenza: 45 patients with laboratory-confirmed influenza and 74 patients whom diagnosis of influenza was made based on clinical and epidemiological data. Umifenovir had influence on the time to resolution of all symptoms. All symptoms were resolved within the first 60 hours after therapy initiation in 23.8% patients with laboratory-confirmed influenza in the umifenovir group and it was 5.7 times greater compared to placebo group (4.2%) (p < 0.05). Severity of illness, catarrhal symptoms and intoxication was reduced with umifenovir compared to placebo, reducing of severity was most evidently observed within the first 2-3 days following the therapy initiation. Umifenovir had a significant effect on viral shedding. The proportion of patients still shedding influenza virus on day 4 was significantly reduced in the umifenovir group compared to placebo (25 vs 53%, respectively; p < 0.05). CONCLUSION: It was found that the effect of umifenovir in the treatment of influenza in adults is most pronounced in the acute stage of the disease and appears in the reduction of time to resolution of all symptoms of the disease, reducing the severity of symptoms of the disease and durations of virus shedding.

Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents.

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

[The use of apoptosis inducers in the therapy of experimental influenza infection and preventing of chronic post-influenza lung damage].

Influenza is a respiratory infection widely spread around the world. Influenza complications are various in nature and in most cases involve the excessive proliferation of cells in respiratory tract as a factor of pathogenesis. In the present work the efficacy of the use of apoptosis inducer 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalenecarboxylic acid (AHPN) for prophylaxis of chronic damage on the stage of post- influenza pneumonia has been studied. Mice were infected with influenza virus A/mallard/Pennsylvania/10218/84(H5N2) with further study of the level of influenza virus reproduction in the lungs, specific mortality of animals and morphology of the foci of post-influenza pneumonia on the 15th day post inoculation. AHPN was shown to decrease the infectious activity of the virus in the lungs by 1.2-1.5 log10 EID50/0.2 mL depending on the dose as compared to the control group, in a weak decrease in mortality of animals (protection index was 12.5-37.5%). The application of AHPN restricted both the proliferative and infiltrative component in chronic post-influenza lesions. It demonstrated the most pronounced effect on the lung morphology when applied on days 4 to 7 post inoculation, i. e. in the period of maximal activation of inflammatory tissue infiltration and regeneration of bronchiolar epithelium. In conclusion, the use of apoptosis inducers can partially prevent the development of chronic post-influenza lesions with proliferative component.

Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins.

Influenza A virus is one of the major human pathogens. Despite numerous efforts to produce absolutely effective anti-influenza drugs or vaccines, no such agent has been developed yet. One of the main reasons for this complication is the high mutation rate and the specific structure of influenza A viruses genome. For more than 25 years since the first mapping of the viral genome, it was believed that its 8 genome segments encode 10 proteins. However, the proteome of influenza A viruses has turned out to be much more complex than previously thought. In 2001, the first accessory protein, PB1-F2, translated from the alternative open reading frame, was discovered. Subsequently, six more proteins, PB1-N40, PA-X, PA-N155, PA-N182, M42, and NS3, have been found. It is important to pay close attention to these novel proteins in order to evaluate their role in the pathogenesis of influenza, especially in the case of outbreaks of human infections with new avian viruses, such as H5N1 or H7N9. In this review we summarize the data on the molecular mechanisms used by influenza A viruses to expand their proteome and on the possible functions of the recently discovered viral proteins.

[Intranasal co-administration of recombinant streptococcus group B polypeptides and influenza deltaNS1 vaccine].

In the present work, the immunoadjuvant properties of the influenza deltaNS1 vaccine virus after intranasal administration in combination with recombinant GBS polypeptides was tested in mice. According to our data, co-administration of recombinant GBS polypeptides and influenza deltaNS1 vaccine resulted in the increase in the immunogenicity and protective efficacy of bacterial proteins. Combined vaccination with the GBS polypeptides and influenza deltaNS1 vaccine has a potential to be used not only for prophylaxis infections caused by SGB, but also for prevention of the bacterial complications of influenza.

[Deitiforine, an antiinfluenza chemotherapeutic].

The problem of prophylaxis and therapy of influenza infections remains one of the priority goals for medical science and practical health care. The review includes the discussion of antiviral activity of Deitiforine, a Russian chemotherapeutic. The data on the toxicity and the specific activity spectrum in cell cultures, chicken embryos and laboratory animals are presented. The problem of the influenza viruses resistance to cage compounds and in particular to rimantadine and Deitiforine is also discussed.

[Universal diagnostic oligonucleotide microarray for subtyping of human and animal influenza A viruses].

The diagnostic oligonucleotide microarray for subtyping of human and animal influenza A viruses (IAVs) was developed. We proposed a simple method of the fluorescent labeling of genomic segments of all known IAVs subtypes, the composition of the hybridization buffer, as well as the software of the data processing. 48 IAVs strains of different subtypes were analyzed using our microarray. All of them were identified, while 45 of 48 strains were unambiguously subtyped.

[Technological approaches to development of whole-virion inactivated vaccine from recombinant strain against A/H5N1 influenza in the Republic of Kazakhstan].

AIM: Development of technological stages of preparation of experimental influenza whole-virion inactivated adsorbed vaccine based on recombinant influenza virus strains NIBRG-14 and A/Astana/RG/6:2/2009. MATERIALS AND METHODS: 2 recombinant vaccines influenza strains were used in the study--NIBRG-14 and A/Astana/RG/6:2/2009. Purification of native virus-containing allantoic fluid was performed by ion-exchange chromatography. The virus was inactivated by formaldehyde. Merthiolate at concentration of 0.1 mg/ml was added to the vaccine as a preserving substance. Aluminium hydroxide was used as an adjuvant. Harmlessness and immunogenicity (HI) of the constructed preparation are determining. RESULTS: Virus-containing materials from recombinant strains with biological activity of 8.5 - 9.0 lg EID50/cm3 and hemagglutination activity of 1:256 - 1:1024 in chicken embryos were obtained. Optimal inactivation regimen of non-purified suspensions by formaldehyde was established and combined scheme of purification and concentration of influenza virus was selected that provide harmlessness and immunogenicity of experimental samples of inactivated vaccines against highly pathogenic influenza A/H5N1 in experiments in mice. CONCLUSION: The data obtained on quality parameters of intermediate products and final vaccine give evidence on their compliance with normative parameters for whole-virion influenza purified vaccine.

[Molecular-biological properties of the rubella virus strains isolated in St. Petersburg].

In the surveillance of rubella in the northwest region of Russia samples of nasopharyngeal swabs from 37 patients with rubella, which were treated in the 442nd district military hospital named after Z.P. Solovyov in autumn 2007 were screened for the rubella virus using RK-13 cell line, 22 strains of rubella virus were isolated. Gene sequencing of E1 region of rubella virus isolates was carried out. Rubella virus strains isolated in St. Petersburg during the 2007 outbreak belonged to rubella virus genotype 1E. The morphogenesis of RK-13 cells with formation of replication complexes and enveloped virions of rubella virus was shown.

[Effect of different water-soluble forms of the fullerene C60 on the metabolic activity and ultra-structure of cells in culture].

In view of contradictory data on the toxicity of fullerenes for live organisms we studied the effect of water-soluble complexes of C60 with N-polyvivyl-pirrolidone (C60/PVP) and gamma-cyclodextrine (C60/gamma-CD) on MA-104 cells in culture. Both complexes proved to be non-toxic for cultured cells in the dark in wide range of concentrations. Both complexes provoke changes of cellular ultra-structure which reflect the enhancement of metabolic activity. At the same time only the exposition with the complex C60/PVP leads to the essential growth of number and size of mitochondria. However, the effect of two studied water-soluble forms of C60 under intensive UV-irradiation of cells proved to be opposite: C60/PVP had a cyto-protective action while C60/gamma-CD caused a significant growth of photo-toxicity. Possible reasons of the differences in the action of different forms of C60 on living organisms are discussed.

[Effect of Ingavirin on the ultrastructure of the morphogenesis of adenovirus infection in vivo].

The goal of this study was to evaluate the effect of Ingavirin on the morphological features of the foci of adenovirus hepatitis in Syrian hamsters by electron microscopy. The use of the drug was shown to cause a substantial reduction in the rate of destructive processes and inflammatory reactions in the liver, by normalizing its structure at the levels of both tissue and individual hepatocytes. After administration of Ingavirin, the morphogenesis of adenovirus infection in the infected hepatocytes did not differ from that in the controls; however, the infected cells were fewer. The proportion of morphologically inadequate virions in the presence of Ingavirin increased from 35 to 46%. The findings suggest that Ingavirin is an effective drug that has antiviral, anti-inflammatory, and cytoprotective activities in the focus of adenovirus tissue involvement.