Highly Pathogenic Avian Influenza A(H5N1) Virus-Induced Mass Death of Wild Birds, Caspian Sea, Russia, 2022.

In May 2022, we observed a substantial die-off of wild migratory waterbirds on Maliy Zhemchuzhniy Island in the Caspian Sea, Russia. The deaths were caused by highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b virus. Continued surveillance of influenza viruses in wild bird populations is needed to predict virus spread over long distances.

Detection of the Omicron SARS-CoV-2 Lineage and Its BA.1 Variant with Multiplex RT-qPCR.

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.

Highly Pathogenic Avian Influenza A(H5N8) Virus Clade 2.3.4.4b, Western Siberia, Russia, 2020.

Two variants of highly pathogenic avian influenza A(H5N8) virus were detected in dead poultry in Western Siberia, Russia, during August and September 2020. One variant was represented by viruses of clade 2.3.4.4b and the other by a novel reassortant between clade 2.3.4.4b and Eurasian low pathogenicity avian influenza viruses circulating in wild birds.

Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection.

RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82-148) NS RNA region influence NS1 protein expression.

Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.

Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-ß1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-ß dependent, underlie its effects.

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.

Reintroduction of highly pathogenic avian influenza A/H5N8 virus of clade 2.3.4.4. in Russia.

In the spring of 2016, a loss of wild birds was observed during the monitoring of avian influenza virus activity in the Republic of Tyva. That outbreak was caused by influenza H5N8 virus of clade 2.3.4.4. In the fall, viruses of H5N8 clade 2.3.4.4 were propagated in European countries. This paper presents some results of analysis of the virus strains isolated during the spring and fall seasons in 2016 in the Russian Federation. The investigated strains were highly pathogenic for mice, and some of their antigenic and genetic features differed from those of an H5N8 strain that circulated in 2014 in Russia.

The Development of the SARS-CoV-2 Epidemic in Different Regions of Siberia in the 2020-2022 Period.

The comparison of the development of the SARS-CoV-2 epidemic in several neighboring regions can help researchers to assess the risks and develop more effective strategies and approaches in the field of preventive medicine. We analyzed the infection and mortality statistics for the 2020-2022 period in ten individual regions of the Siberian Federal District of Russia. We also sequenced complete genomes, which allowed us to analyze the genetic diversity of SARS-CoV-2 circulated in each of the ten regions and to build a phylogenetic dendrogram for the virus variants. The ParSeq v.1.0 software was developed to automate and speed up the processing and analysis of viral genomes. At the beginning of the pandemic, in the first two waves, the B.1.1 variant (20B) dominated in all regions of the Siberian Federal District. The third and fourth waves were caused by the Delta variant. Mortality during this period was at a maximum; the incidence was quite high, but the number of deposited genomes with GISAID during this period was extremely low. The maximum incidence was at the beginning of 2022, which corresponds to the arrival of the Omicron variant in the region. The BA.5.2 variant became the dominant one. In addition, by using NextClade, we identified three recombinants in the most densely populated areas.

Virological and Genetic Characterization of the Unusual Avian Influenza H14Nx Viruses in the Northern Asia.

Wild aquatic birds are generally identified as a natural reservoir of avian influenza viruses (AIVs), where a high diversity of subtypes has been detected. Some AIV subtypes are considered to have relatively low prevalence in wild bird populations. Six-year AIV surveillance in Siberia revealed sporadic cases of the rarely identified H14-subtype AIV circulation. Complete genome sequencing of three H14 isolates were performed, and the analysis indicated interconnections between low pathogenic avian influenza (LPAI) viruses. We conducted hemagglutination inhibition and virus neutralization assays, estimated the susceptibility of isolates to neuraminidase inhibitors, and characterized receptor specificity. Our study revealed circulation of a new H14N9 subtype described for the first time. However, the low prevalence of the H14-subtype AIV population may be the reason for the underestimation of the diversity of H14-subtype AIVs. According to the available data, a region in which H14-subtype viruses were detected several times in 2007-2022 in the Eastern Hemisphere is Western Siberia, while the virus was also detected once in South Asia (Pakistan). Phylogenetic analysis of HA segment sequences revealed the circulation of two clades of H14-subtype viruses originated from initial 1980s Eurasian clade; the first was detected in Northern America and the second in Eurasia.

An Early SARS-CoV-2 Omicron Outbreak in a Dormitory in Saint Petersburg, Russia.

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.

Assessing the Intense Influenza A(H1N1)pdm09 Epidemic and Vaccine Effectiveness in the Post-COVID Season in the Russian Federation.

The COVID-19 pandemic had a profound impact on influenza activity worldwide. However, as the pandemic progressed, influenza activity resumed. Here, we describe the influenza epidemic of high intensity of the 2022-2023 season. The epidemic had an early start and peaked in week 51.2022. The extremely high intensity of the epidemic may have been due to a significant decrease in herd immunity. The results of PCR-testing of 220,067 clinical samples revealed that the influenza A(H1N1)pdm09 virus dominated, causing 56.4% of positive cases, while A(H3N2) influenza subtype accounted for only 0.6%, and influenza B of Victoria lineage-for 34.3%. The influenza vaccine was found to be highly effective, with an estimated effectiveness of 92.7% in preventing admission with laboratory-confirmed influenza severe acute respiratory illness (SARI) cases and 54.7% in preventing influenza-like illness/acute respiratory illness (ILI/ARI) cases due to antigenic matching of circulated viruses with influenza vaccine strains for the season. Full genome next-generation sequencing of 1723 influenza A(H1N1)pdm09 viruses showed that all of them fell within clade 6B.1A.5.a2; nine of them possessed H275Y substitution in the NA gene, a genetic marker of oseltamivir resistance. Influenza A(H3N2) viruses belonged to subclade 3C.2a1b.2a.2 with the genetic group 2b being dominant. All 433 influenza B viruses belonged to subclade V1A.3a.2 encoding HA1 substitutions A127T, P144L, and K203R, which could be further divided into two subgroups. None of the influenza A(H3N2) and B viruses sequenced had markers of resistance to NA inhibitors. Thus, despite the continuing circulation of Omicron descendant lineages, influenza activity has resumed in full force, raising concerns about the intensity of fore coming seasonal epidemics.

SARS-CoV-2 escape from cytotoxic T cells during long-term COVID-19.

Evolution of SARS-CoV-2 in immunocompromised hosts may result in novel variants with changed properties. While escape from humoral immunity certainly contributes to intra-host evolution, escape from cellular immunity is poorly understood. Here, we report a case of long-term COVID-19 in an immunocompromised patient with non-Hodgkin's lymphoma who received treatment with rituximab and lacked neutralizing antibodies. Over the 318 days of the disease, the SARS-CoV-2 genome gained a total of 40 changes, 34 of which were present by the end of the study period. Among the acquired mutations, 12 reduced or prevented the binding of known immunogenic SARS-CoV-2 HLA class I antigens. By experimentally assessing the effect of a subset of the escape mutations, we show that they resulted in a loss of as much as ~1% of effector CD8 T cell response. Our results indicate that CD8 T cell escape represents a major underappreciated contributor to SARS-CoV-2 evolution in humans.

Resurgence of Influenza Circulation in the Russian Federation during the Delta and Omicron COVID-19 Era.

Influenza circulation was substantially reduced after March 2020 in the European region and globally due to the wide introduction of non-pharmaceutical interventions (NPIs) against COVID-19. The virus, however, has been actively circulating in natural reservoirs. In summer 2021, NPIs were loosened in Russia, and influenza activity resumed shortly thereafter. Here, we summarize the epidemiological and virological data on the influenza epidemic in Russia in 2021-2022 obtained by the two National Influenza Centers. We demonstrate that the commonly used baseline for acute respiratory infection (ARI) is no longer sufficiently sensitive and BL for ILI incidence was more specific for early recognition of the epidemic. We also present the results of PCR detection of influenza, SARS-CoV-2 and other respiratory viruses as well as antigenic and genetic analysis of influenza viruses. Influenza A(H3N2) prevailed this season with influenza B being detected at low levels at the end of the epidemic. The majority of A(H3N2) viruses were antigenically and genetically homogenous and belonged to the clade 3C.2a1b.2a.2 of the vaccine strain A/Darwin/9/2021 for the season 2022-2023. All influenza B viruses belonged to the Victoria lineage and were similar to the influenza B/Austria/1359417/2021 virus. No influenza A(H1N1)pdm09 and influenza B/Yamagata lineage was isolated last season.

Respiratory Syncytial Virus G Protein Sequence Variability among Isolates from St. Petersburg, Russia, during the 2013-2014 Epidemic Season.

Human respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. It is actively evolving under environmental and herd immunity influences. This work presents, for the first time, sequence variability analysis of RSV G gene and G protein using St. Petersburg (Russia) isolates. Viruses were isolated in a cell culture from the clinical samples of 61 children hospitalized (January-April 2014) with laboratory-confirmed RSV infection. Real-time RT-PCR data showed that 56 isolates (91.8%) belonged to RSV-A and 5 isolates (8.2%) belonged to RSV-B. The G genes were sequenced for 27 RSV-A isolates and all of them belonged to genotype ON1/GA2. Of these RSV-A, 77.8% belonged to the ON1(1.1) genetic sub-cluster, and 14.8% belonged to the ON1(1.2) sub-cluster. The ON1(1.3) sub-cluster constituted a minor group (3.7%). Many single-amino acid substitutions were identified in the G proteins of St. Petersburg isolates, compared with the Canadian ON1/GA2 reference virus (ON67-1210A). Most of the amino acid replacements were found in immunodominant B- and T-cell antigenic determinants of G protein. These may affect the antigenic characteristics of RSV and influence the host antiviral immune response to currently circulating viruses.

Antigenic and Genetic Characterization of Swine Influenza Viruses Identified in the European Region of Russia, 2014-2020.

Pigs have long been recognized as "mixing vessels" in which new viruses are formed by reassortment involving various influenza virus lineages (avian, animal, human). However, surveillance of swine influenza viruses only gained real significance after the 2009 pandemic. A fundamentally important point is the fact that there is still no regular surveillance of swine flu in Russia, and the role of swine viruses is underestimated since, as a rule, they do not cause serious disease in animals. Since the pig population in Russia is large, it is obvious that the lack of monitoring and insufficient study of swine influenza evolution constitutes a gap in animal influenza surveillance, not only for Russia, but globally. A 6 year joint effort enabled identification of SIV subtypes that circulate in the pig population of Russia's European geographic region. The swine influenza viruses isolated were antigenically and genetically diverse. Some were similar to human influenza viruses of A(H1N1)pdm09 and A(H3N2) subtype, while others were reassortant A(H1pdm09N2) and A(H1avN2) and were antigenically distinct from human H1N1 and H1N1pdm09 strains. Analysis of swine serum samples collected throughout the seasons showed that the number of sera positive for influenza viruses has increased in recent years. This indicates that swine populations are highly susceptible to infection with human influenza viruses. It also stresses the need for regular SIV surveillance, monitoring of viral evolution, and strengthening of pandemic preparedness.

Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs.

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1-124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

Genomic epidemiology of the early stages of the SARS-CoV-2 outbreak in Russia.

The ongoing pandemic of SARS-CoV-2 presents novel challenges and opportunities for the use of phylogenetics to understand and control its spread. Here, we analyze the emergence of SARS-CoV-2 in Russia in March and April 2020. Combining phylogeographic analysis with travel history data, we estimate that the sampled viral diversity has originated from at least 67 closely timed introductions into Russia, mostly in late February to early March. All but one of these introductions were not from China, suggesting that border closure with China has helped delay establishment of SARS-CoV-2 in Russia. These introductions resulted in at least 9 distinct Russian lineages corresponding to domestic transmission. A notable transmission cluster corresponded to a nosocomial outbreak at the Vreden hospital in Saint Petersburg; phylodynamic analysis of this cluster reveals multiple (2-3) introductions each giving rise to a large number of cases, with a high initial effective reproduction number of 3.0 [1.9, 4.3].

Comparative Immunological Study in Mice of Inactivated Influenza Vaccines Used in the Russian Immunization Program.

A series of commercial inactivated influenza vaccines (IIVs) used in the Russian National Immunization Program were characterized to evaluate their protective properties on an animal model. Standard methods for quantifying immune response, such as hemagglutination inhibition (HAI) assay and virus neutralization (VN) assay, allowed us to distinguish the immunogenic effect of various IIVs from that of placebo. However, these standard approaches are not suitable to determine the role of various vaccine components in immune response maturation. The expanded methodological base including an enzyme-linked immunosorbent assay (ELISA) and a neuraminidase ELISA (NA-ELISA) helped us to get wider characteristics and identify the effectiveness of various commercial vaccines depending on the antigen content. Investigations conducted showed that among the IIVs tested, Ultrix®, Ultrix® Quadri and VAXIGRIP® elicit the most balanced immune response, including a good NA response. For Ultrix®, Ultrix® Quadri, and SOVIGRIPP® (FORT LLC), the whole-virus specific antibody subclass IgG1, measured in ELISA, seriously prevailed over IgG2a, while, for VAXIGRIP® and SOVIGRIPP® (NPO Microgen JSC) preparations, the calculated IgG1/IgG2a ratio was close to 1. So, the immune response varied drastically across different commercial IIVs injected in mice.

Experimental Infection Using Mouse-Adapted Influenza B Virus in a Mouse Model.

Every year, influenza B viruses (IBVs) contribute to annual illness, and infection can lead to serious respiratory disease among humans. More attention is needed in several areas, such as increasing virulence or pathogenicity of circulating B viruses and developing vaccines against current influenza. Since preclinical trials of anti-influenza drugs are mainly conducted in mice, we developed an appropriate infection model, using an antigenically-relevant IBV strain, for furtherance of anti-influenza drug testing and influenza vaccine protective efficacy analysis. A Victoria lineage (clade 1A) IBV was serially passaged 17 times in BALB/c mice, and adaptive amino acid substitutions were found in hemagglutinin (HA) (T214I) and neuraminidase (NA) (D432N). By electron microscopy, spherical and elliptical IBV forms were noted. Light microscopy showed that mouse-adapted IBVs caused influenza pneumonia on day 6 post inoculation. We evaluated the illness pathogenicity, viral load, and histopathological features of mouse-adapted IBVs and estimated anti-influenza drugs and vaccine efficiency in vitro and in vivo. Assessment of an investigational anti-influenza drug (oseltamivir ethoxysuccinate) and an influenza vaccine (Ultrix®, SPBNIIVS, Saint Petersburg, Russia) showed effectiveness against the mouse-adapted influenza B virus.

Pathology of A(H5N8) (Clade 2.3.4.4) Virus in Experimentally Infected Chickens and Mice.

The emergence of novel highly pathogenic avian influenza viruses (HPAIVs) in migratory birds raises serious concerns as these viruses have the potential to spread during fall migration. We report the identification of novel HPAIV A(H5N8) clade 2.3.4.4 virus that was isolated from sick domestic duck at commercial farm during the second wave of spread that began in October and affected poultry (ducks; chiсkens) in several European regions of Russia and Western Siberia in 2016. The strain was highly lethal in experimental infection of chickens and mice with IVPI = 2.34 and MLD50 = 1.3log10⁡ EID50, accordingly. Inoculation of chickens with the HPAIV A/H5N8 demonstrated neuroinvasiveness, multiorgan failure, and death of chickens on the 3rd day post inoculation. Virus replicated in all collected organ samples in high viral titers with the highest titer in the brain (6.75±0.1 log10TCID50/ml). Effective virus replication was found in the following cells: neurons and glial cells of a brain; alveolar cells and macrophages of lungs; epithelial cells of a small intestine; hepatocytes and Kupffer cells of a liver; macrophages and endothelial cells of a spleen; and the tubular epithelial cells of kidneys. These findings advance our understanding of histopathological effect of A(H5N8) HPAIV infection.