Phenotypic assessment of antiviral activity for spiro-annulated oxepanes and azepenes.

Evolutionary potential of viruses can result in outbreaks of well-known viruses and emergence of novel ones. Pharmacological methods of intervening the reproduction of various less popular, but not less important viruses are not available, as well as the spectrum of antiviral activity for most known compounds. In the framework of chemical biology paradigm, characterization of antiviral activity spectrum of new compounds allows to extend the antiviral chemical space and provides new important structure-activity relationships for data-driven drug discovery. Here we present a primary assessment of antiviral activity of spiro-annulated derivatives of seven-membered heterocycles, oxepane and azepane, in phenotypic assays against viruses with different genomes, virion structures, and genome realization schemes: orthoflavivirus (tick-borne encephalitis virus, TBEV), enteroviruses (poliovirus, enterovirus A71, echovirus 30), adenovirus (human adenovirus C5), hantavirus (Puumala virus). Hit compounds inhibited reproduction of adenovirus C5, the only DNA virus in the studied set, in the yield reduction assay, and did not inhibit reproduction of RNA viruses.

Non-Polio Enteroviruses Isolated by Acute Flaccid Paralysis Surveillance Laboratories in the Russian Federation in 1998-2021: Distinct Epidemiological Features of Types.

More than 100 types of non-polio enteroviruses (NPEVs) are ubiquitous in the human population and cause a variety of symptoms ranging from very mild to meningitis and acute flaccid paralysis (AFP). Much of the information regarding diverse pathogenic properties of NPEVs comes from the surveillance of poliovirus, which also yields NPEV. The analysis of 265 NPEV isolations from 10,433 AFP cases over 24 years of surveillance and more than 2500 NPEV findings in patients without severe neurological lesions suggests that types EV-A71, E13, and E25 were significantly associated with AFP. EV-A71 was also significantly more common among AFP patients who had fever at the onset and residual paralysis compared to all AFP cases. In addition, a significant disparity was noticed between types that were common in humans (CV-A2, CVA9, EV-A71, E9, and E30) or in sewage (CVA7, E3, E7, E11, E12, and E19). Therefore, there is significant evidence of non-polio viruses being implicated in severe neurological lesions, but further multicenter studies using uniform methodology are needed for a definitive conclusion.

Genomic analysis of Leishmania turanica strains from different regions of Central Asia.

The evolution in Leishmania is governed by the opposite forces of clonality and sexual reproduction, with vicariance being an important factor. As such, Leishmania spp. populations may be monospecific or mixed. Leishmania turanica in Central Asia is a good model to compare these two types. In most areas, populations of L. turanica are mixed with L. gerbilli and L. major. Notably, co-infection with L. turanica in great gerbils helps L. major to withstand a break in the transmission cycle. Conversely, the populations of L. turanica in Mongolia are monospecific and geographically isolated. In this work, we compare genomes of several well-characterized strains of L. turanica originated from monospecific and mixed populations in Central Asia in order to shed light on genetic factors, which may drive evolution of these parasites in different settings. Our results illustrate that evolutionary differences between mixed and monospecific populations of L. turanica are not dramatic. On the level of large-scale genomic rearrangements, we confirmed that different genomic loci and different types of rearrangements may differentiate strains originated from mixed and monospecific populations, with genome translocations being the most prominent example. Our data suggests that L. turanica has a significantly higher level of chromosomal copy number variation between the strains compared to its sister species L. major with only one supernumerary chromosome. This suggests that L. turanica (in contrast to L. major) is in the active phase of evolutionary adaptation.

Patterns and Temporal Dynamics of Natural Recombination in Noroviruses.

Noroviruses infect a wide range of mammals and are the major cause of gastroenteritis in humans. Recombination at the junction of ORF1 encoding nonstructural proteins and ORF2 encoding major capsid protein VP1 is a well-known feature of noroviruses. Using all available complete norovirus sequences, we systematically analyzed patterns of natural recombination in the genus Norovirus both throughout the genome and across the genogroups. Recombination events between nonstructural (ORF1) and structural genomic regions (ORF2 and ORF3) were found in all analyzed genogroups of noroviruses, although recombination was most prominent between members of GII, the most common genogroup that infects humans. The half-life times of recombinant forms (clades without evidence of recombination) of human GI and GII noroviruses were 10.4 and 8.4-11.3 years, respectively. There was evidence of many recent recombination events, and most noroviruses that differed by more than 18% of nucleotide sequence were recombinant relative to each other. However, there were no distinct recombination events between viruses that differed by over 42% in ORF2/3, consistent with the absence of systematic recombination between different genogroups. The few inter-genogroup recombination events most likely occurred between ancient viruses before they diverged into contemporary genogroups. The recombination events within ORF1 or between ORF2/3 were generally rare. Thus, noroviruses routinely exchange full structural and nonstructural blocks of the genome, providing a modular evolution.

Revisiting epidemiology of leishmaniasis in central Asia: lessons learnt.

In this work we reviewed historical and recent data on Leishmania spp. infection combining data collected in Turkmenistan, Uzbekistan, Kazakhstan, Kyrgyzstan, Iran, China and Mongolia. We specifically focused on a complex of co-existing species (Leishmania major, Leishmania turanica and Leishmania gerbilli) sharing the same animal reservoirs and vectors. In addition, we analysed the presence of dsRNA viruses in these species and discussed future research directions to identify species-specific traits, which may determine susceptibility of different Leishmania spp. to viral infection.

Robust AAV Genotyping Based on Genetic Distances in Rep Gene That Are Maintained by Ubiquitous Recombination.

Adeno-associated viruses (AAVs) are a convenient tool for gene therapy delivery. According to the current classification, they are divided into the species AAV A and AAV B within the genus Dependoparvovirus. Historically AAVs were also subdivided on the intraspecies level into 13 serotypes, which differ in tissue tropism and targeted gene delivery capacity. Serotype, however, is not a universal taxonomic category, and their assignment is not always robust. Cross-reactivity has been shown, indicating that classification could not rely on the results of serological tests alone. Moreover, since the isolation of AAV4, all subsequent AAVs were subdivided into serotypes based primarily on genetic differences and phylogenetic reconstructions. An increased interest in the use of AAV as a gene delivery tool justifies the need to improve the existing classification. Here, we suggest genotype-based AAV classification below the species level based on the rep gene. A robust threshold was established as 10% nt differences within the 1248 nt genome fragment, with 4 distinct AAV genotypes identified. This distinct sub-species structure is maintained by ubiquitous recombination within, but not between, rep genes of the suggested genotypes.

Cases of Acute Flaccid Paralysis Associated with Coxsackievirus A2: Findings of a 20-Year Surveillance in the Russian Federation.

Surveillance for acute flaccid paralysis syndrome (AFP) in children under 15 is the backbone of the Global Polio Eradication Initiative. Laboratory examination of stool samples from AFP cases allows the detection of, along with polioviruses, a variety of non-polio enteroviruses (NPEV). The etiological significance of these viruses in the occurrence of AFP cases has been definitively established only for enteroviruses A71 and D68. Enterovirus Coxsackie A2 (CVA2) is most often associated with vesicular pharyngitis and hand, foot and mouth disease. Among 7280 AFP cases registered in Russia over 20 years (2001-2020), CVA2 was isolated only from five cases. However, these included three children aged 3 to 4 years, without overt immune deficiency, immunized with 4-5 doses of poliovirus vaccine in accordance with the National Vaccination Schedule. The disease resulted in persistent residual paralysis. Clinical and laboratory data corresponded to poliomyelitis developing during poliovirus infection. These findings are compatible with CVA2 being the cause of AFP. Molecular analysis of CVA2 from these patients and a number of AFP cases in other countries did not reveal association with a specific phylogenetic group, suggesting that virus genetics is unlikely to explain the pathogenic profile. The overall results highlight the value of AFP surveillance not just for polio control but for studies of uncommon AFP agents.

Analyses of Leishmania-LRV Co-Phylogenetic Patterns and Evolutionary Variability of Viral Proteins.

Leishmania spp. are important pathogens causing a vector-borne disease with a broad range of clinical manifestations from self-healing ulcers to the life-threatening visceral forms. Presence of Leishmania RNA virus (LRV) confers survival advantage to these parasites by suppressing anti-leishmanial immunity in the vertebrate host. The two viral species, LRV1 and LRV2 infect species of the subgenera Viannia and Leishmania, respectively. In this work we investigated co-phylogenetic patterns of leishmaniae and their viruses on a small scale (LRV2 in L. major) and demonstrated their predominant coevolution, occasionally broken by intraspecific host switches. Our analysis of the two viral genes, encoding the capsid and RNA-dependent RNA polymerase (RDRP), revealed them to be under the pressure of purifying selection, which was considerably stronger for the former gene across the whole tree. The selective pressure also differs between the LRV clades and correlates with the frequency of interspecific host switches. In addition, using experimental (capsid) and predicted (RDRP) models we demonstrated that the evolutionary variability across the structure is strikingly different in these two viral proteins.

Citizen science initiative points at childhood BCG vaccination as a risk factor for COVID-19.

Current results do not provide conclusive evidence on the effect of BCG vaccination on COVID-19 alone or in combination with other factors. To address this limitation, in this study we used a citizen science initiative on the COVID-19 pandemic to collect data worldwide during 2 October 2020-30 October 2020 (1,233 individuals) in a structured way for analysing factors and characteristics of affected individuals in relation to BCG vaccination. For the first time, the results of our study suggested that vaccination with BCG may increase the risk for COVID-19 at certain age, particularly in individuals vaccinated at childhood. Childhood BCG vaccination increased the likelihood of being diagnosed with COVID-19 fivefold in COVID-19 low-incidence countries and threefold in high-incidence countries. A reasonable explanation for this effect is the activation of certain innate immunity mechanisms associated with inflammatory reactions. These factors should be considered when analysing the risks associated with this global pandemic.

A hepatitis B virus causes chronic infections in equids worldwide.

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions.

Bats host many viruses pathogenic to humans, and increasing evidence suggests that rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America, and Africa were PCR-positive for RVA, and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, which included evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite various levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses.IMPORTANCE The increased research on bat coronaviruses after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) allowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general, and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents sheds light on the vast genetic diversity of rotaviruses and also hints at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

Evaluating the Impact of Anthropogenic Factors on the Dissemination of Contemporary Cosmopolitan, Arctic, and Arctic-like Rabies Viruses.

Rabies is a globally prevalent viral zoonosis that causes 59,000 deaths per year and has important economic consequences. Most virus spread is associated with the migration of its primary hosts. Anthropogenic dissemination, mainly via the transportation of rabid dogs, shaped virus ecology a few hundred years ago and is responsible for several current outbreaks. A systematic analysis of aberrant long-distance events in the steppe and Arctic-like groups of rabies virus was performed using statistical (Bayesian) phylogeography and plots of genetic vs. geographic distances. The two approaches produced similar results but had some significant differences and complemented each other. No phylogeographic analysis could be performed for the Arctic group because polar foxes transfer the virus across the whole circumpolar region at high velocity, and there was no correlation between genetic and geographic distances in this virus group. In the Arctic-like group and the steppe subgroup of the cosmopolitan group, a significant number of known sequences (15-20%) was associated with rapid long-distance transfers, which mainly occurred within Eurasia. Some of these events have been described previously, while others have not been documented. Most of the recent long-distance transfers apparently did not result in establishing the introduced virus, but a few had important implications for the phylogeographic history of rabies. Thus, human-mediated long-distance transmission of the rabies virus remains a significant threat that needs to be addressed.

TBEV Subtyping in Terms of Genetic Distance.

Currently, the lowest formal taxon in virus classification is species; however, unofficial lower-level units are commonly used in everyday work. Tick-borne encephalitis virus (TBEV) is a species of mammalian tick-borne flaviviruses that may cause encephalitis. Many known representatives of TBEV are grouped into subtypes, mostly according to their phylogenetic relationship. However, the emergence of novel sequences could dissolve this phylogenetic grouping; in the absence of strict quantitative criterion, it may be hard to define the borders of the first TBEV taxonomic unit below the species level. In this study, the nucleotide/amino-acid space of all known TBEV sequences was analyzed. Amino-acid sequence p-distances could not reliably distinguish TBEV subtypes. Viruses that differed by less than 10% of nucleotides in the polyprotein-coding gene belonged to the same subtype. At the same time, more divergent viruses were representatives of different subtypes. According to this distance criterion, TBEV species may be divided into seven subtypes: TBEV-Eur, TBEV-Sib, TBEV-FE, TBEV-2871 (TBEV-Ob), TBEV-Him, TBEV-178-79 (TBEV-Bkl-1), and TBEV-886-84 (TBEV-Bkl-2).

Analysis of miRNA Expression in Patients with Rheumatoid Arthritis during Olokizumab Treatment.

Rheumatoid arthritis (RA) is the most common autoimmune disease worldwide. Epigenetic alternations of microRNAs (miRNAs) can contribute to its pathogenesis and progression. As the first line therapy with DMARDs is not always successful, other drugs and therapeutic targets should be applied. This study aims to measure the expression level of plasma miRNAs in RA patients treated with olokizumab and to evaluate their potential as prognostic biomarkers. The expression of 9 miRNAs was quantified in 103 RA patients before treatment and at weeks 12 and 24 of olokizumab therapy by reverse transcription-polymerase chain reaction (RT-PCR) assay and analyzed in groups of responders and non-responders. Almost all miRNAs changed their expression during therapy. The ROC curve analysis of the most prominent of them together with consequent univariate and multivariate regression analysis revealed statistically significant associations with the olokizumab therapy efficiency scores for miR-26b, miR-29, miR-451, and miR-522. Therefore, these miRNAs might be a potential therapeutic response biomarker.

Fungal Enzyme l-Lysine α-Oxidase Affects the Amino Acid Metabolism in the Brain and Decreases the Polyamine Level.

The fungal glycoprotein l-lysine α-oxidase (LO) catalyzes the oxidative deamination of l-lysine (l-lys). LO may be internalized in the intestine and shows antitumor, antibacterial, and antiviral effects in vivo. The main mechanisms of its effects have been shown to be depletion of the essential amino acid l-lys and action of reactive oxidative species produced by the reaction. Here, we report that LO penetrates into the brain and is retained there for up to 48 h after intravenous injection, which might be explained by specific pharmacokinetics. LO actively intervenes in amino acid metabolism in the brain. The most significant impact of LO was towards amino acids, which are directly exposed to its action (l-lys, l-orn, l-arg). In addition, the enzyme significantly affected the redistribution of amino acids directly associated with the tricarboxylic acid (TCA) cycle (l-asp and l-glu). We discovered that the depletion of l-orn, the precursor of polyamines (PA), led to a significant and long-term decrease in the concentration of polyamines, which are responsible for regulation of many processes including cell proliferation. Thus, LO may be used to reduce levels of l-lys and PA in the brain.

Baltic Group Tick-Borne Encephalitis Virus Phylogeography: Systemic Inconsistency Pattern between Genetic and Geographic Distances.

Tick-Borne Encephalitis Virus (TBEV) is a dangerous arbovirus widely distributed in Northern Eurasia. The area of this pathogen changes over time. At the beginning of the 2000s, the Ixodes tick populations in Karelia increased. At the same time, the area of I. persulcatus, the main vector of the Siberian TBEV subtype, also expanded. Herein, we sequenced 10 viruses isolated from ticks collected in three locations from the Karelia region in 2008-2018. PCR positive samples were passaged in suckling mice or pig embryo kidney cells (PEK). After the second passage in suckling, mice viral RNA was isolated and E-gene fragment was sequenced. Viral sequences were expected to be similar or nearly identical. Instead, there was up to a 4.8% difference in nucleotide sequence, comparable with the most diverse viruses belonging to the Baltic subgroup in Siberian TBEV subtype (Baltic TBEV-Sib). To reveal whether this was systemic or incidental, a comprehensive phylogeographical analysis was conducted. Interestingly, viruses within each geographic region demonstrated comparable diversity to the whole Baltic TBEV-Sib. Moreover, Baltic TBEV-Sib has a distribution area limited by three ecological regions. This means that active virus mixing occurs in the vast geographic area forming one common virus pool. The most plausible explanation is the involvement of flying animals in the TBEV spread.

Phlebovirus sequences detected in ticks collected in Russia: Novel phleboviruses, distinguishing criteria and high tick specificity.

Phlebovirus is an abundant and rather heterogeneous genus within the Phenuiviridae family (order Bunyavirales). The genus Phlebovirus is divided into two antigenic complexes, which also correspond to the main vector: sandflies/mosquitoes and ticks. Previously, only sandfly/mosquito-borne phleboviruses were associated with human disease, such as Rift Valley fever virus, Toscana virus, Sicilian and Naples Sandfly fever viruses and others. Until recently, tick-borne phleboviruses were not considered as human pathogens. After the discovery of severe fever with thrombocytopenia syndrome, interest to tick-borne phleboviruses has increased dramatically. In the last decade, many novel phleboviruses have been reported in different regions. Despite this, the diversity, ecology and pathogenicity of these viruses still remain obscure. The aim of this work was to study the diversity of phleboviruses in ticks collected in several regions of Russia. We used pan-phlebovirus RT-PCR assays based on multiple degenerate primers targeting the polymerase gene fragment. Arthropod specimens were collected from 2005 to 2018. A total of 5901 Ixodidae ticks combined into 1116 pools were screened. A total of 160 specific amplicons were produced. In three cases RT-PCR assays amplified two distinct viruses from same tick pools. Direct sequencing of amplicons and subsequent phylogenetic analysis revealed twelve representatives of divergent phlebovirus groups. Based on the distribution of pairwise nucleotide sequence identity values, a cut-off (88%) was suggested to distinguish tick-borne phleboviruses. According to this provisional criterion, two viruses found here could be termed novel, while ten viruses have been described in previous studies. Detected phleboviruses demonstrated almost perfect specificity to a tick species or, at least, a genus. The same pattern was observed for tick-borne phleboviruses found in different studies around the world. Viruses that grouped together on a phylogenetic tree and differed less than this sequence identity threshold suggested above were hosted by ticks from the same genus.

Genetic Polymorphisms Associated with Rheumatoid Arthritis Development and Antirheumatic Therapy Response.

Rheumatoid arthritis (RA) is the most common inflammatory arthropathy worldwide. Possible manifestations of RA can be represented by a wide variability of symptoms, clinical forms, and course options. This multifactorial disease is triggered by a genetic predisposition and environmental factors. Both clinical and genealogical studies have demonstrated disease case accumulation in families. Revealing the impact of candidate gene missense variants on the disease course elucidates understanding of RA molecular pathogenesis. A multivariate genomewide association study (GWAS) based analysis identified the genes and signalling pathways involved in the pathogenesis of the disease. However, these identified RA candidate gene variants only explain 30% of familial disease cases. The genetic causes for a significant proportion of familial RA have not been determined until now. Therefore, it is important to identify RA risk groups in different populations, as well as the possible prognostic value of some genetic variants for disease development, progression, and treatment. Our review has two purposes. First, to summarise the data on RA candidate genes and the increased disease risk associated with these alleles in various populations. Second, to describe how the genetic variants can be used in the selection of drugs for the treatment of RA.

Seroprevalence and incidence of human toxocarosis in Russia.

Overall incidence of toxocariasis in Russia is low and varies between 1.6 and 2.7 per 100,000, while in several hyper-endemic regions, such as Altay, Kurgan, Perm and Udmurtia, it reaches 43 per 100,000. The seroprevalence of toxocariasis in published references was on average 16% and varied across the regions of Russia from negligible in North Siberia to 40% in southern regions of West Siberia. Seroprevalence in adults in five regions of Russia identified in this study was on average 20%, and varied from 3% in Yakutia (north of East Siberia) to 36% in Rostov-on-Don, South Russia. There was no correlation between seroprevalence and reported incidence of toxocariasis; however, the pattern of seroprevalence variation could be linked to Toxocara prevalence in dogs. Toxocariasis seroprevalence has more than doubled over the last 20 years. Diagnostic antibody titres (1:800 or more) were found in 3.6% of sera, suggesting about five million of acute Toxocara invasions per year.

Toxocara prevalence in dogs, cats and the environment in Russia.

The toxocariasis incidence in Russia is relatively low (1.8 cases per 100,000 individuals) and the parasite is not a major healthcare concern. However, the proportion of primary hosts testing positive for the parasite is high and varies between 3% and 100% in dogs (on average 33%), and between 6% and 52% in cats. Higher prevalence was observed in Volga, Urals and Siberia regions. Levels of contamination of soil, children's playgrounds and sandboxes is also high, with up to 100% contamination rates determined in some studies, but more commonly prevalence of contamination around 40% was reported. There is a pronounced seasonality in the prevalence of Toxocara in primary hosts and the soil, with peaks in the summer and autumn. Most likely, a lack of permissive conditions for the development of eggs in the winter determines observed seasonal patterns, which are different than those observed in most other countries. Toxocara eggs were found in 4-10% of vegetables and greenery samples tested, suggesting that they can contribute to the transmission of Toxocara.