New approach of genetic characterization of group A rotaviruses by the nanopore sequencing method.

Nanopore sequencing of virus genomes represented by segmented RNA (e.g. rotaviruses) requires the development of specific approaches. Due to the massive use of rotavirus vaccines, the relevance of monitoring the genetic diversity of circulating strains of group A rotaviruses (RVA) increased. The WHO recommended method of multiplex type-specific PCR does not allow genotyping of all clinically significant strains of RVA and identifying inter-strain differences within the genotype. We have described a new principle of amplification of RVA gene segments using six primers for reverse transcription and one universal primer for PCR for nanopore sequencing. The amplification of RVA genome was tested on clinical samples and three phylogenetically distant laboratory RVA strains, Wa (G1P[8]), DS-1 (G2P[4]) and 568 (G3P[3]). The developed protocol of sample preparation and nanopore sequencing allowed obtaining full-length sequences for gene segments of RVA, including the diagnostically significant segments 9 (VP7), 4 (VP4) and 6 (VP6) with high accuracy and coverage. The accuracy of sequencing of the rotavirus genome exceeded 99.5 %, and the genome coverage varied for different strains from 59.0 to 99.6 % (on average 86 %). The developed approach of nanopore sequencing of RVA genome could be a prospective tool for epidemiological studies and surveillance of rotavirus infection.

Molecular-Genetic Characterization of Human Rotavirus A Strains Circulating in Moscow, Russia (2009-2014).

Enteric viruses are the most common cause of acute gastroenteritis (AGE) in young children and a significant public health problem globally. Hospital admissions of children under 5 years of age with diarrhea are primarily associated with group A rotavirus (RVA) infection. In this retrospective study, the population structure of viruses linked to AGE etiology in young children hospitalized with AGE in Moscow was evaluated, and molecular characterization of RVA strains was performed. Fecal specimens were collected from children under 5 years old hospitalized with AGE between 2009 and 2014 in Moscow, Russia. Multiplex real-time reverse transcription PCR was used to detect enteric viruses and for G/[P]-genotyping of isolated RVAs. Sequencing of RVA VP7 and VP4 cDNA fragments was used to validate the data obtained by PCR-genotyping. The main causes for hospitalization of children with AGE were RVA (40.1%), followed by noroviruses (11.4%), while adenoviruses, astroviruses, sapoviruses, enteroviruses, and orthoreoviruses were detected in 4.7%, 1.9%, 1.4%, 1.2%, and 0.2% of samples tested, respectively. Nosocomial infections, predominantly associated with RVAs and noroviruses, were detected in 24.8% of cases and occurred significantly more frequently in younger infants. The predominant RVA genotype was G4P[8], detected in 38.7% of RVA-positive cases, whereas genotypes G1P[8], G9P[8], G3P[8], and G2P[4] were found in 11.8%, 6.6%, 4.2%, and 3.3% of cases, respectively. Together, the presence of circulating RVA strains with rare VP7 and VP4 gene variants (G6 and P[9]) highlights the need to conduct continuous epidemiological monitoring of RVA infection.

Water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride as a nucleic acids vector for cell transfection.

To endow the cationic polysaccharides with solubility in the whole pH-range without loss of functionality of the amino groups, different chitosan samples were treated with glycidyltrimethylammonium chloride. Each modified unit of the exhaustively alkylated quaternized chitosan (QCht) contained both quaternary and secondary amino groups. The intercalated dye displacement assay and ζ-potential measurements implied stability of QCht polyplexes at physiological conditions and protonation of the secondary amino groups in slightly acidic media which is favorable for transfection according to proton sponge mechanism. The cytotoxicity and transfection efficacy increased with the chain lengthening. Nevertheless, the longest chains of QCht, 250 kDa were less toxic than PEI for COS-1 cells and revealed comparable and even significantly higher transfection activity of siRNA and plasmid DNA, respectively. Thus, highly polymerized QCht (250 kDa) provided the highest level of the plasmid DNA transfection being 5 and 80 times more active than QCht (100 kDa) and QCht (50 kDa), respectively, and 4-fold more effective than PEI, 25 kDa. The established influence of QCht molecular weight on toxicity and transfection efficacy allows elaborating polysaccharide vectors that possess rational balance of these characteristics.