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Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Human adenovirus 7 (hAdv7) 19BOVLB/Volgograd/Rus/2014 was isolated from the autopsy material from an adult with fatal pneumonia in Volgograd, Russia, in March 2014. Whole-genome sequencing of the virus isolate was performed., (Copyright © 2016 Yatsyshina et al.)

Aim: Establish genetic characteristics, carry out phylogenetic analysis and determination of molecular markers of resistance to etiotropic preparations against influenza A/H3N2 and B viruses that had circulated in Russia in 2013 - 2015., Materials and Methods: 80 biological samples containing influenza A/H3N2 virus RNA and 31 samples containing influenza B virus RNA were studied. Sequencing of PCR fragments was carried out inABI-3 100 PRIZMTM GeneticAnalyzer (AppliedBiosystems, USA) and using MiSeq (Illumina, USA). Data treatment and analysis was carried out using CLC v.3.6.5., DNASTAR and BioNumerics v.6.5. programs., Results: In 2013 -2014 A/Texas/50/2012-like-clade 3C.3 influenza A/H3N2 viruses dominated, 10% belonged to subclade 3C.2a and 10% - to 3C.3b. Most of the viruses (8 1%) of 2014 - 2015 were of 3C.2a clade, the portion of viruses belonging to 3C.3b and 3C.3a was 9 and 10%. Yamagata-like viruses predominated among the studied influenza B viruses, only 1 virus of 2014 - 2015 belonged to Victoria lineage, 1 reassortant of Yamagata and Victoria lineages was detected. Rimantadine- resistance mutationS3 lN(M2 protein) was detected in all the influenza A/H3N2 viruses. Mutations determining resistance to oseltamivir (NA gene) were not detected in influenza A/H3N2 and B viruses., Conclusion: Increase of influenza morbidity in 2014 - 2015 was determined by the emergence of influenza A/H3N2 and B viruses, antigenically distinct from those that had circulated previously and those included into the vaccine, thus resulting in the WHO decision to change A/ H3N2 and B components of the 2015 - 2016 vaccine: Simultaneous circulation of 2 lineages of influenza B virus and emergence of their reassortants gives evidence on the necessity of use of quadrivalent vaccines, containing both lineages.