Whole genome sequencing (WGS) is considered the best instrument to track both virus evolution and the spread of new, emerging variants. However, WGS still does not allow the analysis of as many samples as qPCR does. Epidemiological and clinical research needs to develop advanced qPCR methods to identify emerging variants of SARS-CoV-2 while collecting data on their spreading in a faster and cheaper way, which is critical for introducing public health measures. This study aimed at designing a one-step RT-qPCR assay for multiplex detection of the Omicron lineage and providing additional data on its subvariants in clinical samples. The RT-qPCR assay demonstrated high sensitivity and specificity on multiple SARS-CoV-2 variants and was cross-validated by WGS.

Background/Objectives: Intranasal vaccination enhances protection against respiratory viruses by providing stimuli to the immune system at the primary site of infection, promoting a balanced and effective response. Influenza vectors with truncated NS1 are a promising vaccine approach that ensures a pronounced local CD8+ T-cellular immune response. Here, we describe the protective and immunomodulating properties of an influenza vector FluVec-N carrying the C-terminal fragment of the SARS-CoV-2 nucleoprotein within a truncated NS1 open reading frame. Methods: We generated several FluVec-N recombinant vectors by reverse genetics and confirmed the vector’s genetic stability, antigen expression in vitro, attenuation, and immunogenicity in a mouse model. We tested the protective potential of FluVec-N intranasal immunization in naïve mice and seropositive Th2-prone mice, primed with aluminium-adjuvanted inactivated SARS-CoV-2. Immune response in immunized and challenged mice was analyzed through serological methods and flow cytometry. Results: Double intranasal immunization of naïve mice with FluVec-N reduced weight loss and viral load in the lungs following infection with the SARS-CoV-2 beta variant. Mice primed with alum-adjuvanted inactivated coronavirus experienced substantial early weight loss and eosinophilia in the lungs during infection, demonstrating signs of enhanced disease. A single intranasal boost immunization with FluVec-N prevented the disease enhancement in primed mice by modulating the local immune response. Protection was associated with the formation of specific IgA and the early activation of virus-specific effector and resident CD8+ lymphocytes in mouse lungs. Conclusions: Our study supports the potential of immunization with influenza vector vaccines to prevent respiratory diseases and associated immunopathology.

Human adenoviruses (HAdVs) are globally distributed pathogens capable of causing a wide range of clinical manifestations, particularly acute respiratory infections. However, their genomic diversity remains insufficiently characterized, with substantial geographic gaps in available sequence data, including for Russia, where only a few complete genomes have been deposited prior to this work. In this study, we analyzed more than 1200 PCR-positive respiratory specimens collected from hospitalized patients within routine surveillance projects and the Global Influenza Hospital Surveillance Network (GIHSN) across plenty of Russian regions during 2023–2024. Virus isolation followed by next-generation sequencing yielded 128 complete HAdV genomes representing species B, C, and D. The dataset included 27 B3, 9 B7, 44 B55, 12 C1, 16 C2, 4 C5, 7 C89, 5 C108, and one D109 genome, as well as three unassigned recombinant viruses with p89h5f5, p5h6f6 and p5h57f6 genomic structures (p, penton base; h, hexon; f, fiber). Phylogenetic analyses of whole genomes and capsid genes revealed extensive variability in immunogenic regions, particularly in species C, and identified clusters within B3 viruses. Notably, HAdV-D109 was identified in Russia, marking only the second reported detection of this genotype worldwide. Together, these findings substantially expand the currently available genomic landscape of HAdVs, highlighting the circulation of diverse and recombinant strains in Russia.

On 26 November 2021 WHO designated a new variant of concern B.1.1.529 named Omicron. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Implementation of the high-throughput rRT-PCR screening for Omicron is of great importance for monitoring the spread of this VOC in the population, especially in resource-limited countries lacking sufficient sequencing capacity. Omicron lineage B.1.1.529 (BA.1) has some indels that turned out to be a good target for its detection. In the current protocol, we use ins214EPE for this purpose. Here we describe the 1-step quantitative multiplex RT-qPCR assay consisting of the newly developed Ins214EPE detection set and widely used Hong Kong University N gene assay for SARS-CoV-2 detection (Chu et al., 2020). The assay was validated on the Omicron variant RNA kindly provided by the Pathogenic Microorganisms Variability Laboratory (Dr. Vladimir Guschin, Gamaleya Institute, Moscow, Russia) and RNA from the collection of Smorodintsev Research Institute of Influenza. Omicron RNA specimens were positive in the assay as expected. Negative controls were found negative. 10-fold serial dilutions of Omicron RNA were used to assess ins214EPE assay amplification efficiency. The amplification efficiency was 98,9% (R2 = 0,99). The developed rRT-PCR assay demonstrates high specificity. It was tested on 26 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 8 different SARS-CoV-2 lineages (including Delta B.1.617.2+AY.*) Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza, parainfluenza, human seasonal coronaviruses (OC43, NL63, 229E, HKU1), hRSV, rhinoviruses, bocaviruses, metapneumovirus (33 in total) - with no false-positive results. Ins214EPE Cq 6x B.1.1.7 2x B.1.351 5x AT.1 6x B.1.617.2 4x AY.122 P.1 B.1.1.529 28,72 B.1.1.529 26,29 virus RP Cq SARS Cq Ins214 Cq RSV A 28,76 RSV A 30,56 RSV A 27,70 RSV B 31,49 RSV B 30,98 RSV B 32,33 NL63 32,20 NL63 30,42 NL63 24,95 Oc43 30,34 Oc43 30,69 Oc43 28,64 HKU1 30,06 HKU1 28,30 HKU1 30,73 229E 29,11 229E 32,52 229E 29,37 BoV 32,26 BoV 30,75 BoV 27,25 Rv 32,85 Rv 33,76 Rv 27,75 Piv1 28,63 Piv2 24,72 Piv3 27,01 Piv4 23,90 Adv 29,47 MPV 30,12 HIV A 29,13 HIV A 28,45 HIV A 28,16 39,06 c+ 34,15 26,61 28,44 Analytical sensitivity determination is underway. We consider developed assay to be useful in wide populational RT-PCR screening to assess the spread of Omicron variant.

The Omicron variant of SARS-CoV-2 rapidly spread worldwide in late 2021–early 2022, displacing the previously prevalent Delta variant. Before 16 December 2021, community transmission had already been observed in tens of countries globally. However, in Russia, the majority of reported cases at that time had been sporadic and associated with travel. Here, we report an Omicron outbreak at a student dormitory in Saint Petersburg between 16–29 December 2021, which was the earliest known instance of a large-scale community transmission in Russia. Out of the 465 sampled residents of the dormitory, 180 (38.7%) tested PCR-positive. Among the 118 residents for whom the variant had been tested by whole-genome sequencing, 111 (94.1%) were found to carry the Omicron variant. Among these 111 residents, 60 (54.1%) were vaccinated or had reported a previous infection of COVID-19. Phylogenetic analysis confirmed that the outbreak was caused by a single introduction of the BA.1.1 sub-lineage of the Omicron variant. The dormitory-derived clade constituted a significant proportion of BA.1.1 samples in Saint Petersburg and has spread to other regions of Russia and even to other countries. The rapid spread of the Omicron variant in a population with preexisting immunity to previous variants underlines its propensity for immune evasion.

Background/Objectives: The current strategy for seasonal influenza prophylaxis relies on updating the vaccine components annually to account for the rapid antigenic drift of viruses and the low cross-protective efficacy of available vaccines. Mutant influenza viruses with truncated or deleted NS1 protein are known to stimulate cross-specific T-cell immune response and provide protection against heterosubtypic influenza A and B viruses. Methods: We generated NS1ΔC influenza A and B viruses with C-terminal NS1 deletions by reverse genetics. In a mouse model, we assessed the safety and immunogenicity of the B/Lee/NS1ΔC strain upon intranasal administration, as well as the mechanism of its cross-protective efficacy against sublethal B/Victoria and B/Yamagata challenges. We then investigated the potential of the intranasal Flu/NS1ΔC vaccine–a trivalent formulation of NS1ΔC A/H1N1, A/H3N2, and B influenza viruses–to protect mice from lethal influenza infection with homologous, heterologous, and antigenically drifted influenza A and B viruses. Results: Intranasal immunization with the B/Lee/NS1ΔC strain was safe in mice. It activated cross-specific T-cell responses in the lungs and protected animals against heterologous challenge by reducing viral load, inflammation, and lung pathology. Immunization with the trivalent Flu/NS1ΔC vaccine formulation improved survival and reduced weight loss and viral load upon challenge with A/H1N1pdm, A/H2N2, A/H5N1, and B/Victoria viruses. Conclusions: The trivalent intranasal Flu/NS1ΔC influenza vaccine is a promising tool to improve seasonal influenza protection and preparedness for an influenza pandemic.