Soil Giant Phage: Genome and Biological Characteristics of Sinorhizobium Jumbo Phage.

This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage-microbe interactions with nitrogen-fixing symbiotic microorganisms.

CRISPR-Cas9 mediated knockout of AnxA6 gene enhances influenza A virus replication in low-permissive HEK293FT cell line.

Using cell cultures of human origin for the propagation of influenza virus is an attractive way to preserve its glycosylation profile and antigenic properties, which is essential in influenza surveillance and vaccine production. However, only few cell lines are highly permissive to influenza virus, and none of them are of human origin. The barrier might be associated with host restriction factors inhibiting influenza growth, such as AnxA6 protein counteracting the process of influenza virion packaging. In the presented work we explore the CRISPR-Cas9 mediated knockout of ANXA6 gene as a way to overcome the host restriction barrier and increase the susceptibility of human cell line to influenza infection. By CRISPR-Cas9 genome editing we modified HEK293FT cells and obtained several clones defective in the ANXA6 gene. The replication of the influenza A virus in original HEK293FT cells and the HEK293FT-ANXA6-/- mutant cells was compared in growth curve experiments. By combination of methods including TCID assay and flow cytometry we showed that accumulation of influenza A virus in the mutant HEK293FT-ANXA6-/- cells significantly exceeded the virus titer in the original HEK293FT cells.

Clean and folded: Production of active, high quality recombinant human interferon-λ1.

Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-λ1) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-λ1 was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-λ1 were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to feature expected specific biological activity in line with published effects: induction of MxA gene expression in A549 cells and antiviral activity against influenza A virus.

Safety and efficacy of p62 DNA vaccine ELENAGEN in a first-in-human trial in patients with advanced solid tumors.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy.

Development of a realtime RT-PCR assay for the rapid detection of influenza A(H2) viruses.

Influenza and other acute respiratory infections are of great concern for public health, causing excessive morbidity and mortality throughout the world. Influenza virus A(H2N2), which caused a pandemic of so called "Asian flu" in 1957 was expelled from the human population by the new pandemic virus subtype H3N2 in 1968, however, influenza A(H2) viruses continue to circulate in wild birds and poultry. The lack of immunity in human population and the continued circulation of influenza A(H2) among animals makes emergence of a new pandemic virus possible. One of the basic techniques of molecular diagnostics of infectious diseases is the realtime polymerase chain reaction (PCR). The aim of this work was to design oligonucleotide primers and probes for the rapid detection of influenza A virus subtype H2 by realtime reverse transcription - polymerase chain reaction (rRT-PCR). Analysis of 539 sequences of influenza A(H2N2) virus hemagglutinin gene from GISAID EpiFlu database revealed conservative regions suitable for use as binding sites for primers and probes. 191 probes were designed and 2 sets of primers and probes (H2-1 and H2-2) were selected for further experimental evaluation. Detection limit of RT-PCR system was 50 copies of DNA per 25 µl reaction when 10-fold dilutions of pCI-neo-H2 plasmid used as template. Analytical specificity of selected sets of primers and probes were tested on wide range of influenza strains and non-influenza respiratory viruses. H2-2 set found to have insufficient specificity detecting seasonal influenza A(H1N1) viruses and was excluded from further analysis. Analytical sensitivity was further tested on vaccine strain A/17/California/66/395 (H2N2) and A/Japan/305/1957 (H2N2), limit of detection for primers-probe set H2-1 was 3.2 (CI95%: 3.07-3.48) lg EID50/ml. Designed primers and probes for the realtime RT-PCR universal detection of influenza A(H2) viruses could be used in clinical trials of vaccines against influenza A(H2) and screening for H2 in cases of unsubtypeable influenza A in humans.

Genetic characterization of influenza viruses from influenza-related hospital admissions in the St. Petersburg and Valencia sites of the Global Influenza Hospital Surveillance Network during the 2013/14 influenza season.

BACKGROUND: Continuous surveillance for genetic changes in circulating influenza viruses is needed to guide influenza prevention and control. OBJECTIVES: To compare intra-seasonal influenza genetic diversity of hemagglutinin in influenza A strains isolated from influenza hospital admissions collected at two distinct sites during the same season. STUDY DESIGN: Comparative phylogenetic analysis of full-length hemagglutinin genes from 77 isolated influenza A viruses from the St. Petersburg, Russian Federation and Valencia, Spain sites of the Global Influenza Hospital Surveillance Network (GIHSN) during the 2013/14 season. RESULTS: We found significant variability in A(H3N2) and A(H1N1)pdm09 viruses between the two sites, with nucleotide variation at antigenic positions much lower for A(H1N1)pdm09 than for A(H3N2) viruses. For A(H1N1)pdm09, antigenic sites differed by three to four amino acids from the vaccine strain, two of them common to all tested isolates. For A(H3N2) viruses, antigenic sites differed by six to nine amino acids from the vaccine strain, four of them common to all tested isolates. A fifth amino acid substitution in the antigenic sites of A(H3N2) defined a new clade, 3C.2. For both influenza A subtypes, pairwise amino acid distances between circulating viruses and vaccine strains were significantly higher at antigenic than at non-antigenic sites. Whereas A(H1N1)pdm09 viruses clustered with clade 6B and 94% of A(H3N2) with clade 3C.3, at both study sites A(H3N2) clade 3C.2 viruses emerged towards the end of the season, showing greater pairwise amino acid distances from the vaccine strain compared to the predominant clade 3C.3. CONCLUSIONS: Influenza A antigenic variants differed between St. Petersburg and Valencia, and A(H3N2) clade 3C.2 viruses were characterized by more amino acid differences from the vaccine strain, especially at the antigenic sites.

Rapid spread of influenza A(H1N1)pdm09 viruses with a new set of specific mutations in the internal genes in the beginning of 2015/2016 epidemic season in Moscow and Saint Petersburg (Russian Federation).

A dramatic increase of influenza activity in Russia since week 3 of 2016 significantly differs from previous seasons in terms of the incidence of influenza and acute respiratory infection (ARI) and in number of lethal cases. We performed antigenic analysis of 108 and whole-genome sequencing of 77 influenza A(H1N1)pdm09 viruses from Moscow and Saint Petersburg. Most of the viruses were antigenically related to the vaccine strain. Whole-genome analysis revealed a composition of specific mutations in the internal genes (D2E and M83I in NEP, E125D in NS1, M105T in NP, Q208K in M1, and N204S in PA-X) that probably emerged before the beginning of 2015/2016 epidemic season.

Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector.

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors - B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.

INFLUENZA SURVEILLANCE IN RUSSIA BASED ON EPIDEMIOLOGICAL AND LABORATORY DATA FOR THE PERIOD FROM 2005 TO 2012.

Exchange of information on and sharing of influenza viruses through the GISRS network has great significance for understanding influenza virus evolution, recognition of a new pandemic virus emergence and for preparing annual WHO recommendations on influenza vaccine strain composition. Influenza surveillance in Russia is based on collaboration of two NICs with 59 Regional Bases. Most epidemiological and laboratory data are entered through the internet into the electronic database at the Research Institute of Influenza (RII), where they are analyzed and then reported to the Ministry of Public Health of Russia. Simultaneously, data are introduced into WHO's Flu Net and Euro Flu, both electronic databases. Annual influenza epidemics of moderate intensity were registered during four pre-pandemic seasons. Children aged 0-2 and 3-6 years were the most affected groups of the population. Influenza registered clinically among hospitalized patients with respiratory infections for the whole epidemic period varied between 1.3 and 5.4% and up but to 18.5-23.0% during the peak of the two pandemic waves caused by influenza A(H1N1) pdm 09 virus and to lesser extent (2.9 to 8.5%) during usual seasonal epidemics. Most epidemics were associated with influenza A(H1N1), A(H3N2) and B co-circulation. During the two pandemic waves (in 2009-2010 and 2010-2011) influenza A(H1N1) pdm 09 predominated. It was accompanied by a rapid growth of influenza morbidity with a significant increase of both hospitalization and mortality. The new pandemic virus displaced the previous seasonal A(H1N1) virus completely. As a rule, most of the influenza viruses circulating in Russia were antigenic ally related to the strains recommended by WHO for vaccine composition for the Northern hemisphere with the exception of two seasons when an unexpected replacement of the influenza B Victoria lineage by Yamagata lineage (2007-2008) and the following return of Victoria lineage viruses (2008-2009) was registered. Influenza surveillance in Russia was improved as a result of enhancing capacity to international standards and the introduction of new methods in NICs such as rRT-PCR diagnosis, regular testing of influenza viruses for susceptibility to antivirals, phylogenetic analysis as well as organization of sentinel surveillance in a number of Regional Base Laboratories. Improvements promoted rapid recognition of the appearance a new pandemic virus in the country and enhancement of confirmation tests in investigation of influenza related death cases.