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.
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 EID/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.
Antigenic and genetic characteristics of Russian RSV isolates are presented for the first time. Of the 69 strains isolated in St. Petersburg, 93% belonged to the RSV-A antigenic group. The antigenic variations in the F-protein RSV were analyzed using a panel from 6 monoclonal antibodies by the method of micro-cultural ELISA. Depending on the decrease in the effectiveness of interaction with monoclonal antibodies (relative to the reference strain Long), RSV-A isolates were divided into 4 antigenic subgroups. The results of 24 isolates sequencing showed that more than 60% of them had substitutions in significant F-protein sites compared to the ON67-1210A reference strain of the current RSV genotype ON1/GA2. The most variable were the signal peptide and antigenic site II. When comparing the results of ELISA and sequencing, it was not possible to identify any specific key substitutions in the amino acid sequence of the F-protein that affect the interaction of the virus with antibodies. The nucleotide sequence of the F-gene from 19 of the 24 characterized isolates was close to that of ON67-1210A reference virus and was significantly different from RSV-A Long and A2 viruses. A separate group consisted of 5 strains, in which the F-protein structure was approximated to RSV Long.
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