Mammarenavirus guanaritoense (formerly Guanarito virus, GTOV) is an important etiological agent of an emergent disease with a high mortality rate, namely Venezuelan hemorrhagic fever (VHF). Traditionally, VHF is an important public health problem and is a mandatory reportable disease. Moreover, global interconnection has increased the probability of worldwide spread of GTOV. Therefore, national healthcare organizations need tools for the management of GTOV-associated risks. These tools should definitely include diagnostic kits based on real-time reverse transcription PCR (RT-PCR) because it is the most suitable method for the diagnosis of viral hemorrhagic fever diseases. Here, we describe a real-time RT-PCR assay for the detection of GTOV. This assay was developed and evaluated using armored positive control particles (ARC). The LOD of the assay is in the range 1–2.5 × 102 RNA copies/mL on the CFX96 PCR plate machine and is in the range 1–2.0 × 103 RNA copies/mL on the Rotor-Gene Q PCR rotary machine. Our assay was evaluated using positive GTOV samples from the collection of the Instituto Nacional de Higiene “Rafael Rangel”, Caracas, Venezuela (INHRR). The assay provides a fast and sensitive tool for GTOV detection. The high specificity and sensitivity of the assay make it useful for clinical and epidemiological investigations in the field of VHF and their etiological agents.

Lake Baikal, one of the oldest lakes on Earth and the deepest freshwater lake in the world, harbors unique viral communities shaped by its ancient, oligotrophic, and highly oxygenated ecosystem. In this study, the first comprehensive analysis of circular Rep-encoding single-stranded (CRESS) DNA viruses in the plankton fraction (<0.2 μm) of Lake Baikal is presented. Metagenomic sequencing identified 47 CRESS-like genomes (1003–5249 nucleotides), including 12 putative complete genomes, encoding replication (Rep) and capsid proteins, with limited similarity to known sequences in RefSeq database (maximum identity: 77.2 %). Phylogenetic analysis revealed that the Rep proteins from Lake Baikal cluster with unclassified CRESS groups (CRESS1, CRESS2, CRESS3, CRESS5) and Nanoviridae/Alphasatellitidae, form neighboring clusters with Circoviridae and CRESS4, and form the individual “Baikal” clusters distinct from established virus families. These clusters suggest long-term diversification in isolation, potentially linked to endemic hosts or gaps in the global CRESS virus sampling. Genome organization predominantly followed Types II and V (ambisense/unisense), with conserved nonanucleotide motifs (e.g., TAGTATTAC) and Rep domains typical of CRESS viruses. Notably, no identical matches were found in databases, emphasizing the viromic novelty of Baikal. The occurrence of some virus genomes across years, seasons, and depths highlights that they are ecologically widespread in Lake Baikal. Our findings expand the known diversity of CRESS DNA viruses and emphasize the role of ancient lakes as reservoirs of undescribed viral lineages, driven by unique evolutionary pressures.

Ticks of the genus Ixodes are recognized as important vectors of a wide range of viral pathogens with potential implications for public and veterinary health. Recent advances in metagenomic sequencing have uncovered an unprecedented diversity within tick-associated viromes, yet much of the global tick metavirome remains unexplored, particularly in vast and ecologically diverse regions such as Northwestern Russia. In this study, we present a comprehensive metaviromic and phylogenetic characterization of viruses detected in Ixodes persulcatus and Ixodes ricinus ticks collected from five regions in Northwestern Russia between 2021 and 2023. Using high-throughput RNA sequencing, we identified viral sequences representing families Nairoviridae, Partitiviridae, Phenuiviridae, Flaviviridae, Chuviridae, and Narnaviridae, Orthototiviridae. Putative novel viral lineages were identified. Phylogenetic analyses revealed strong geographic structuring of some viral lineages. This suggests either the presence of local genotypes, or underrepresentation of Eurasian tick-associated viromes, in current databases. In addition to TBEV, other viruses previously associated with human illness were detected in ticks in Northwestern Russia (Beiji nairovirus, Mukawa virus). Our findings provide the first high-resolution snapshot of the tick virome in Northwestern Russia. They emphasize the importance of continued viral surveillance in underrepresented biogeographic zones. These data contribute to the growing global virome map and may inform the development of region-specific vector-borne disease countermeasures.

Among the highly oncogenic human papillomavirus (HPV) types, HPV 16 is of paramount importance in the pathogenesis of cervical cancer. Viral oncogenes E6 and E7, expressed by the HPV genome, play a key role in developing neoplasia. The aim was to study the prevalence, mutations in the E6–E7 genes, HPV 16 lineages and sublineages in underlying, precancerous lesions, and cervical cancer. Materials and methods. Cervical samples from 223 patients with morphologically confirmed background, precancerous diseases and cervical cancer in St. Petersburg were examined. HPV detection and genotyping were performed using real-time PCR. Sequencing of the E6–E7 genes of HPV 16 was performed using the Sanger method. Single-nucleotide polymorphisms were identified against the European lineage HPV 16 K02718 reference sequence. Results. In patients with underlying conditions, the proportion of HPV-positive samples was 55.0%. As the severity of tumor lesions progressed, the frequency of viral detection increased: from 65.0% in low-grade dysplasia to 95.1% in squamous cell carcinoma (p = 0.002). Monoinfection with HPV 16 was identified in 68.3% of patients with precancerous and malignant cervical lesions. The proportion of HPV 16 increased with the progression of neoplasia: from 38.4% in low-grade dysplasia to 77.6% in cervical cancer (p = 0.008). Sequencing of HPV 16 E6–E7 genes revealed nucleotide variations characteristic of phylogenetic lineage A. The most prevalent variant was 350G (56.3%), whose detection rate in cervical cancer exceeded that in the group without pathology — 69.8% vs 44.2% (p = 0.019). The frequency of the European variant 350T ranged from 9.7% to 35.5% depending on the degree of cervical lesion. Phylogenetic analysis revealed that 98.6% of clinical isolates belonged to lineage A, of which 74.8% were assigned to sublineage A2 of HPV 16. Sublineages A1, A2, A3, and D1 were detected in background cervical diseases, with a predominance of sublineage A2 (p = 0.001). Multiple mutations in the E6–E7 genes were detected in two isolates from imported cases, and they were assigned to sublineage D1. Only sublineages A1 and A2 of HPV 16 were detected in cervical intraepithelial neoplasia and cervical cancer. Conclusion. The study results showed that in a megacity in northwestern Russia, HPV 16 sublineage A2 plays a leading role in the etiopathogenesis of precancerous lesions and cervical cancer, with sublineage A1 also making a substantial contribution.

Introduction. Continuous monitoring of the epidemiological situation is one of the key elements of a country's biological safety. The development and increasing accessibility of high-throughput sequencing technologies have led to the incorporation of genomic data into public health decision-making systems, known as genomic epidemiological surveillance. Effective implementation of genomic epidemiological surveillance for circulating pathogens requires finding a balance between data generation speed, estimation accuracy, and rational use of resources. Consequently, there is a need to develop evidence-based strategies for biological sample selection. Aim: To justify the choice of methods for calculating the minimum sufficient sample size for sequencing depending on the objectives of epidemiological surveillance. Materials and methods. This study presents a comparative analysis of two approaches to genomic monitoring: routine surveillance and targeted sentinel surveillance. The choice of statistical approaches for calculating the minimum sufficient sample size for each objective is justified, with specific examples that allow determining the required sequencing volume taking into account the current epidemiological situation and the prevalence of pathogen variants in the population. Results. The study presents sample size calculation results depending on the objective: establishing the proportion of existing variants for routine surveillance, or detecting rare variants within sentinel surveillance at a predetermined prevalence threshold for a rare/new variant with a specified probability. For routine surveillance, the sample size was calculated based on the requirement for estimation precision of proportions (margin of error 5%) at a confidence level of 95%. For sentinel surveillance, the sample size was calculated under the condition that the probability of missing a variant (not detecting a single case) does not exceed 5%. The statistical results are presented in the form of graphs and calculation tables. Conclusion. The proposed methods for calculating sample sizes for genomic monitoring depending on the objective can serve as a practical tool for epidemiological surveillance, increasing its efficiency. The use of these calculation methods will make it possible to obtain mathematically sound results regarding the genotypes of circulating pathogens and their degree of representation in the population.

Russia is a country with a high incidence of tick-borne encephalitis (TBE). In northwestern regions of Russia, 110 TBE cases were registered in 2021. The largest numbers of TBE cases were registered in the Arkhangelsk region and St. Petersburg. TBEV seropositivity among healthy individuals, including the unvaccinated population in northwestern Russia, was found in 12.2% of studied samples, indicating active TBEV circulation. The prevalence of TBEV is 2.4% in the two tick species most common in northwestern regions of Russia, Ixodes ricinus and Ixodes persulcatus. However, there is still no comprehensive data on the molecular characterization and phylogenetic analysis of the circulating TBEV strains. The purpose of the study was to determine the prevalence of TBEV and to identify its subtypes in ixodid ticks collected in specific areas of northwestern Russian regions. Phylogenetic analysis of E protein sequences of ten obtained strains showed that they all belong to the Siberian subtype, which were clustered into two groups: the most numerous Baltic group, clusteron 3D; and the Vasilchenko group. However, some unique isolates may form new clusterons.

Phleboviruses, a diverse group within the Phenuiviridae family, include mosquito-, tick-, and sandfly-borne viruses, several of which are pathogenic to humans and animals. Among them, Phlebovirus mukawaense (formerly Mukawa virus, MKWV), a tick-associated member of the Phlebovirus genus, exhibits features, indicative of an emerging arbovirus with potential pathogenicity for humans and animals. This study investigated the distribution, genetic diversity, and host/vector interactions of MKWV across Russia. It identified two genotypes: the previously described Mukawa genotype and the newly characterized Gomselga genotype. This study presents complete genomic sequences of ten MKWV isolates from Russia and analyzes their phylogenetic relationship with other known isolates. The isolates share 88-89% nucleotide identity, and 97-98% RdRp amino acid identity, with the MKWV reference strain (MKW73). This places them within the same species under current ICTV classification criteria. Genetic distance correlated with geographic origin for the L and M segments. The S segment, particularly the NSs gene fragment, showed less regional variability. The findings suggest that previously described Gomselga viruses in Russia are part of the MKWV species and highlight the virus's broader geographic distribution and genetic diversity across East Asia and Europe. Segment reassortments were observed both within the Gomselga genotype and within the Mukawa genotype. While the Mukawa genotype replicates in mammalian cells, the Gomselga genotype does not. This indicates possible differences in host tropism or pathogenic potential. Despite these differences, both genotypes have evolutionary features consistent with arboviruses, including reassortment and geographical clustering of genomic variants. Although vertical transmission in ticks remains unproven, the detection of MKWV in multiple tick species and vertebrate hosts supports the likelihood of vector-borne transmission. These findings suggest that MKWV is a probable arboviral pathogen with an extensive geographic range and potential public health relevance. This warrants further surveillance and virological characterization to assess its epidemic potential.

Arboviruses, including but not limited to dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), pose a significant global threat to human health. The transmission of DENV, ZIKV, and CHIKV is facilitated by mosquitoes belonging to the genus Aedes, which are prevalent in both urban and rural regions of Vietnam. In 2023 an investigation into the population of mosquitoes was conducted in a number of provinces located within the central region of Vietnam. A total of 12,546 mosquitoes were collected during the study. The mosquitoes collected comprised the genera Culex spp., Aedes spp., Anopheles spp., and Armigeres spp. The Aedes spp. mosquitoes were predominant, being collected in 908 pools. These were then examined by RT-qPCR for the detection of DENV, ZIKV, and CHIKV. DENV viral RNA was detected in 92 mosquito pools, ZIKV was detected in 1 mosquito pool, and CHIKV was not detected. The typing of samples containing DENV RNA was carried out. It is evident from the results of the typing process that three distinct types of DENV have been identified. The three main dengue virus types are DENV-1, DENV-2, and DENV-4.

The severity of MeV infection has been greatly reduced by the development of a live attenuated vaccine, which has been incorporated into vaccination programs in many countries. However, poor access to health facilities, and above all, the increase in anti-vaccination movements, has prevented the achievement of sufficient vaccination coverage. In outbreak scenarios, a rapid and transportable method can improve differential diagnosis, including removing ambiguity in suspected measles cases, contacts, or a cohort. In response to the need, we have developed a new RT-qPCR-based MeV detection assay. The LOD of the developed assay was determined on different PCR machines and the higher threshold was 1-1.2 103 copies/mL. The joint diagnostic sensitivity of ELISA and RT-PCR (used together) was 100%, and used combinedly, these two methods enable detection of all measles-infected persons, which is extremely important for controlling contagion and spread of infection. During the clinical validation of the assay on 200 clinical samples from measles-suspected cases using ELISA, 157 samples showed a positive result, while 163 positive cases were confirmed by the RT-qPCR assay. The concordance between the two techniques was 93%. According to our results, the real-time RT-PCR approach used in our study is more sensitive and appears to be a more promising method for measles diagnosis during early stages of the disease, likely before the rise of specific IgM antibodies detected by ELISA.

IntroductionBats are considered the natural reservoirs of several viruses including coronaviruses (CoVs), two genera of which, alpha- and betacoronaviruses, infect humans. Despite widespread screening of bat samples for novel viruses, information on the diversity of coronaviruses in bats inhabiting Russian territory remains scarce. Here, we analyzed the presence and diversity of alphacoronaviruses (Alpha-CoVs) in bats from European Russia.MethodsFourty three fecal samples from bats of 8 species: P. nathusii, P. kuhlii, M. brandtii, M. daubentonii, N. noctula, V. murinus, M. dasycneme, and P. auritus were taken to study.Results and discussionWe detected Alpha-CoV RdRp gene fragments in 30% (13/43) of samples examined in 75% (6/8) of species sampled. Phylogenetic analysis of RdRp showed that most of the identified Alpha-CoV sequences fall into clades within the Pedacovirus subgenus, with minor clusters of nyctacoviruses or myotacoviruses. We assert that closely related pedacoviruses have been circulating for a long time (from 2015 to 2021) in a large region from European Russia to Northern Europe. We propose that closely related pedacoviruses collected from common areas represent a separate species, which we name NE-Alpha coronavirus, with its host being bats of the genus Pipistrellus that inhabit a region from the European part of Russia to Northern Europe. Among the animals sampled, 4.6% (2/43) carried two Alpha-CoVs related to different subgenera (pedacovirus/myotacovirus or pedacovirus/nyctacovirus) simultaneously. We confirmed the presence of two different Alpha-CoV subgenera related to pedacovirus and nyctacovirus in P. kuhlii captured in 2021 and kept in captivity using whole genome sequencing of these viruses. The presence of two or more coronaviruses in one individual animal host is an essential prerequisite for recombination to occur. We also obtained two Alpha-CoV whole genomes from two specimens of P. nathusii captured in 2015. The genomic organization of BatCoV/MOW15-21 and BatCoV/MOW15-23 was similar to other Alpha-CoVs, but the assembled genomes contained a long insertion in the ORF1ab gene which has not been described in other Alpha-CoVs, except for a single sequence from P. nathusii captured in the Netherlands. We propose that the insertion encodes a previously undescribed domain of unknown function, probably related to the SEA domain superfamily.

Enteroviruses (EV) are important pathogens causing human disease with various clinical manifestations. To date, treatment of enteroviral infections is mainly supportive since no vaccination or antiviral drugs are approved for their prevention or treatment. Here, we describe the antiviral properties and mechanisms of action of leucoverdazyls—novel heterocyclic compounds with antioxidant potential. The lead compound, 1a, demonstrated low cytotoxicity along with high antioxidant and virus-inhibiting activity. A viral strain resistant to 1a was selected, and the development of resistance was shown to be accompanied by mutation of virus-specific non-structural protein 2C. This resistant virus had lower fitness when grown in cell culture. Taken together, our results demonstrate high antiviral potential of leucoverdazyls as novel inhibitors of enterovirus replication and support previous evidence of an important role of 2C proteins in EV replication.

The widespread use of the oral poliovaccine from Sabin strains (tOPV) radically reduced poliomyelitis incidence worldwide. However, OPV became a source of neurovirulent vaccine-derived polioviruses (VDPVs). Currently, circulating type 2 VDPVs (cVDPV2) are the leading cause of poliomyelitis. The novel OPV type 2 vaccine (nOPV2), based on genetically modified Sabin strain with increased genetic stability and reduced risk of cVDPV formation, has been used to combat cVDPV2 outbreaks, including one in Tajikistan in 2021. In order to identify the importation of cVDPV2 and nOPV2-derivates, stool samples from 12,127 healthy migrant children under 5 years of age arriving from Tajikistan were examined in Russia (March 2021-April 2022). Viruses were isolated in cell culture and identified via intratype differentiation RT-PCR, VP1 and whole-genome sequencing. cVDPV2 isolates closely related with the Tajikistan one were isolated from two children, and nOPV2-derived viruses were detected in specimens from 106 children from 37 regions of Russia. The duration of nOPV2 excretion ranged from 24 to 124 days post-vaccination. nOPV2 isolates contained 27 mutations per genome (0.36%) on average, with no critical genetic changes, which confirms the genetic stability of nOPV2 during field use. The possibility of epidemiologically significant poliovirus introduction into polio-free countries has been confirmed. The screening of special populations, including migrants, is required to maintain epidemiological well-being.

In aquatic ecosystems, biodegradation of microcystins is the main route of elimination and detoxification. In this study, we discuss the ability of bacterioplankton of lakes Baikal, Doroninskoye, Gusinoye to degrade cyclic microcystin molecule. PCR analysis showed the absence of the key enzyme microcystinase mlrA for mlr-pathway. At the same time, metagenomic analysis of a community of the toxic cyanobacterium Tychonema sp. BBK16 revealed the presence of the mrlC gene responsible for cleavage of microcystin molecule linear residues. In addition, all bacteria-symbionts of cyanobacteria contain glutathione transferases - enzymes involved in nonspecific degradation of lipophilic toxic substances, which also include microcystins.

Based on the success of the Sabin2-based vaccine, a next-generation nOPV2 poliovirus vaccine has been developed. For epidemic monitoring and conducting epidemiological investigations, it is necessary to have a diagnostic assay with the ability to differentiate this variant from others. Here we describe such a real-time RTPCR assay. The region with the cre insertion in the 5 '-UTR was chosen as the target, and the limit of detection was 103 copies/mL (2.5x103 copies/mL using Probit analysis) determined using armored RNA particles. Sensitivity and specificity were 86.28 - 100 % and 76.84 - 100 %, respectively (with 95 % CI). Thus, this method can be effectively used when it is necessary to make a differential diagnosis of poliovirus strains.

Ultra-small bacteria are ubiquitous in the environment, but for some time they have remained out of scientific focus. One of the reasons is their small size. Ultramicrobacteria, which are defined as bacteria with a cell size of less than 0.1 µm3, can pass through a filter with a pore size of 0.2 µm. Moreover, there are some difficulties associated with their cultivation. Currently, relatively pure cultures have been obtained, but mostly for dominant phyla. Rare phyla are represented only by single strains. At the same time, ultramicrobacteria are found in a wide range of environments, and their high-ordered, small genomes and unique physiological features attract the increasing attention of researchers. The development of metagenomics allows us to partially overcome cultivation limitations, and in this article, we present metagenomic assemblies derived from water samples of the largest oligotrophic lakes on the Earth, Baikal (Russian Federation) and Khubsugul (Mongolia). In this study, we carried out the taxonomic and functional analysis of reconstructed genomes of Saccharimonadia class bacteria (phylum Patescibacteria), which is intended to expand our knowledge of rare representatives of microbial communities and supplement the already available information regarding the inhabitants of the oldest ecosystems.

Dugbe virus (DUGV) is a tick-borne arbovirus first isolated in Nigeria in 1964. It has been detected in many African countries using such diverse methods as serological tests, virus isolation, and molecular detection. In Senegal, reports of DUGV isolates mainly occurred in the 1970s and 1980s. Here, we report a contemporary detection of three novel DUGV isolates upon screening of a total of 2877 individual ticks regrouped into 844 pools. The three positive pools were identified as Amblyomma variegatum, the main known vector of DUGV, collected in the southern part of the country (Kolda region). Interestingly, phylogenetic analysis indicates that the newly sequenced isolates are globally related to the previously characterized isolates in West Africa, thus highlighting potentially endemic, unnoticed viral transmission. This study was also an opportunity to develop a rapid and affordable protocol for full-genome sequencing of DUGV using nanopore technology. The results suggest a relatively low mutation rate and relatively conservative evolution of DUGV isolates.

Abstract Background Since its beginnings in 2019, the COVID-19 pandemic is still a problem of global medical concern. Southern Vietnam is one of the country's vast regions, including 20 provinces and the densely populated metropolis Ho Chi Minh City. The study aimed to investigate the epidemiology of COVID-19, including whole-genome sequencing of SARS-CoV-2 samples from Southern Vietnam, during the period January 2020 to December 2021 for identification of the main circulating variants. Methods Epidemiological data were obtained from the Department of Preventive Medicine of the Vietnamese Ministry of Health. To identify circulating variants, RNA, extracted from 126 nasopharyngeal swabs of patients with suspected COVID-19 were sequenced on Illunina MiSeq to obtain near complete genomes SARS-CoV-2. Results Due to the effectiveness of restrictive measures in Vietnam, it was possible to keep incidence at a low level. The partial relaxation of restrictive measures, and the spread of Delta lineages, contributed to the beginning of a logarithmic increase in incidence. Lineages 20A-H circulated in Southern Vietnam during 2020. Spread of the Delta lineage in Southern Vietnam began in March 2021, causing a logarithmic rise in the number of COVID-19 cases. Conclusions Pandemic dynamics in Vietnam feature specific variations in incidence, and these reflect the success of the restrictive measures put in place during the early stages of the pandemic.

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We have studied the Def1 and Def2 genes in order to establish the relationship between the distribution of polymorphism in these genes and the geographical distribution of Apis cerana populations in South Korea, Vietnam, and Russia. Based on the obtained nucleotide sequences, phylogenetic trees were built, which show that the polymorphism of the Def1 and Def2 genes is not associated with geographical distribution of A. cerana populations.

BACKGROUND:Since its beginnings in 2019, the COVID-19 pandemic is still a problem of global medical concern. Southern Vietnam is one of the country's vast regions, including 20 provinces and the densely populated metropolis Ho Chi Minh City. A randomized retrospective study was performed to investigate the epidemiology and genetic diversity of COVID-19. Whole-genome sequencing of 126 SARS-CoV-2 samples collected from Southern Vietnam between January 2020 and December 2021 revealed the main circulating variants and their distribution.METHODS:Epidemiological data were obtained from the Department of Preventive Medicine of the Vietnamese Ministry of Health. To identify circulating variants, RNA, extracted from 126 nasopharyngeal swabs of patients with suspected COVID-19 were sequenced on Illunina MiSeq to obtain near complete genomes SARS-CoV-2.RESULTS:Due to the effectiveness of restrictive measures in Vietnam, it was possible to keep incidence at a low level. The partial relaxation of restrictive measures, and the spread of Delta lineages, contributed to the beginning of a logarithmic increase in incidence. Lineages 20A-H circulated in Southern Vietnam during 2020. Spread of the Delta lineage in Southern Vietnam began in March 2021, causing a logarithmic rise in the number of COVID-19 cases.CONCLUSIONS:Pandemic dynamics in Southern Vietnam feature specific variations in incidence, and these reflect the success of the restrictive measures put in place during the early stages of the pandemic.