IAVCP (Influenza A Virus Consensus and Phylogeny): Automatic Identification of the Genomic Sequence of the Influenza A Virus from High-Throughput Sequencing Data.

Recently, high-throughput sequencing of influenza A viruses has become a routine test. It should be noted that the extremely high diversity of the influenza A virus complicates the task of determining the sequences of all eight genome segments. For a fast and accurate analysis, it is necessary to select the most suitable reference for each segment. At the same time, there is no standardized method in the field of decoding sequencing results that allows the user to update the sequence databases to which the reads obtained by virus sequencing are compared. The IAVCP (influenza A virus consensus and phylogeny) was developed with the goal of automatically analyzing high-throughput sequencing data of influenza A viruses. Its goals include the extraction of a consensus genome directly from paired raw reads. In addition, the pipeline enables the identification of potential reassortment events in the evolutionary history of the virus of interest by analyzing the topological structure of phylogenetic trees that are automatically reconstructed.

Codon-optimization in gene therapy: promises, prospects and challenges.

Codon optimization has evolved to enhance protein expression efficiency by exploiting the genetic code's redundancy, allowing for multiple codon options for a single amino acid. Initially observed in E. coli, optimal codon usage correlates with high gene expression, which has propelled applications expanding from basic research to biopharmaceuticals and vaccine development. The method is especially valuable for adjusting immune responses in gene therapies and has the potenial to create tissue-specific therapies. However, challenges persist, such as the risk of unintended effects on protein function and the complexity of evaluating optimization effectiveness. Despite these issues, codon optimization is crucial in advancing gene therapeutics. This study provides a comprehensive review of the current metrics for codon-optimization, and its practical usage in research and clinical applications, in the context of gene therapy.

Evolutionary Formation and Distribution of Puumala Virus Genome Variants, Russia.

We analyzed Puumala virus (PUUV) sequences collected from bank voles from different regions of Russia. Phylogenetic analysis revealed PUUV reassortments in areas with the highest hemorrhagic fever with renal syndrome incidence, indicating reassortment might contribute to pathogenic properties of PUUV. Continued surveillance is needed to assess PUUV pathogenicity in Russia.

AAV genome modification for efficient AAV production.

The adeno-associated virus (AAV) is one of the most potent vectors in gene therapy. The experimental profile of this vector shows its efficiency and accepted safety, which explains its increased usage by scientists for the research and treatment of a wide range of diseases. These studies require using functional, pure, and high titers of vector particles. In fact, the current knowledge of AAV structure and genome helps improve the scalable production of AAV vectors. In this review, we summarize the latest studies on the optimization of scalable AAV production through modifying the AAV genome or biological processes inside the cell.

Models of Congenital Adrenal Hyperplasia for Gene Therapies Testing.

The adrenal glands are important endocrine organs that play a major role in the stress response. Some adrenal glands abnormalities are treated with hormone replacement therapy, which does not address physiological requirements. Modern technologies make it possible to develop gene therapy drugs that can completely cure diseases caused by mutations in specific genes. Congenital adrenal hyperplasia (CAH) is an example of such a potentially treatable monogenic disease. CAH is an autosomal recessive inherited disease with an overall incidence of 1:9500-1:20,000 newborns. To date, there are several promising drugs for CAH gene therapy. At the same time, it remains unclear how new approaches can be tested, as there are no models for this disease. The present review focuses on modern models for inherited adrenal gland insufficiency and their detailed characterization. In addition, the advantages and disadvantages of various pathological models are discussed, and ways of further development are suggested.

Cap-Independent Circular mRNA Translation Efficiency.

Recently, the mRNA platform has become the method of choice in vaccine development to find new ways to fight infectious diseases. However, this approach has shortcomings, namely that mRNA vaccines require special storage conditions, which makes them less accessible. This instability is due to the fact that the five-prime and three-prime ends of the mRNA are a substrate for the ubiquitous exoribonucleases. To address the problem, circular mRNAs have been proposed for transgene delivery as they lack these ends. Notably, circular RNAs do not have a capped five-prime end, which makes it impossible to initiate translation canonically. In this review, we summarize the current knowledge on cap-independent translation initiation methods and discuss which approaches might be most effective in developing vaccines and other biotechnological products based on circular mRNAs.

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.

Universal Flu mRNA Vaccine: Promises, Prospects, and Problems.

The seasonal flu vaccine is, essentially, the only known way to prevent influenza epidemics. However, this approach has limited efficacy due to the high diversity of influenza viruses. Several techniques could potentially overcome this obstacle. A recent first-in-human study of a chimeric hemagglutinin-based universal influenza virus vaccine demonstrated promising results. The coronavirus pandemic triggered the development of fundamentally new vaccine platforms that have demonstrated their effectiveness in humans. Currently, there are around a dozen messenger RNA and self-amplifying RNA flu vaccines in clinical or preclinical trials. However, the applicability of novel approaches for a universal influenza vaccine creation remains unclear. The current review aims to cover the current state of this problem and to suggest future directions for RNA-based flu vaccine development.

A fatal case of haemorrhagic fever with renal syndrome in Kursk Region, Russia, caused by a novel Puumala virus clade.

Haemorrhagic fever with renal syndrome (HFRS) is the most widespread natural-focal human disease in the Russian Federation. In this study, we report virological assessment of a fatal case of HFRS-PUUV (Puumala virus) in the Kursk Region. The infection caused severe multiorgan failure and the maximum viral load was detected in the tissue of the spleen. Viral sequences were obtained from the patient's autopsy material and lung tissues of bank voles captured in the region. These sequences formed a new clade in the PUUV phylogenetic tree, an outgroup to all known Russian (RUS) lineage sequences. On the other hand viruses collected in the Kursk Region grouped with the RUS lineage and are separated from all other PUUV linages. We propose to nominate this novel group as W-RUS as the identified viruses were collected near the western Russian boundary. The recombination signals between their ancestors and RUS lineage representatives from the Volga region were revealed. The strain Samara_94/CG/2005 suggestively emerged as the result of reassortment between the ancestors of W-RUS and DTK-Ufa-97.

Modular Evolution of Coronavirus Genomes.

The viral family Coronaviridae comprises four genera, termed Alpha-, Beta-, Gamma-, and Deltacoronavirus. Recombination events have been described in many coronaviruses infecting humans and other animals. However, formal analysis of the recombination patterns, both in terms of the involved genome regions and the extent of genetic divergence between partners, are scarce. Common methods of recombination detection based on phylogenetic incongruences (e.g., a phylogenetic compatibility matrix) may fail in cases where too many events diminish the phylogenetic signal. Thus, an approach comparing genetic distances in distinct genome regions (pairwise distance deviation matrix) was set up. In alpha, beta, and delta-coronaviruses, a low incidence of recombination between closely related viruses was evident in all genome regions, but it was more extensive between the spike gene and other genome regions. In contrast, avian gammacoronaviruses recombined extensively and exist as a global cloud of genes with poorly corresponding genetic distances in different parts of the genome. Spike, but not other structural proteins, was most commonly exchanged between coronaviruses. Recombination patterns differed between coronavirus genera and corresponded to the modular structure of the spike: recombination traces were more pronounced between spike domains (N-terminal and C-terminal parts of S1 and S2) than within domains. The variability of possible recombination events and their uneven distribution over the genome suggest that compatibility of genes, rather than mechanistic or ecological limitations, shapes recombination patterns in coronaviruses.

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).

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.

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.

Tick-Borne Encephalitis Virus: An Emerging Ancient Zoonosis?

Tick-borne encephalitis (TBE) is one of the most important viral zoonosis transmitted by the bite of infected ticks. In this study, all tick-borne encephalitis virus (TBEV) E gene sequences available in GenBank as of June 2019 with known date of isolation (n = 551) were analyzed. Simulation studies showed that a sample bias could significantly affect earlier studies, because small TBEV datasets (n = 50) produced non-overlapping intervals for evolutionary rate estimates. An apparent lack of a temporal signal in TBEV, in general, was found, precluding molecular clock analysis of all TBEV subtypes in one dataset. Within all subtypes and most of the smaller groups in these subtypes, there was evidence of many medium- and long-distance virus transfers. These multiple random events may play a key role in the virus spreading. For some groups, virus diversity within one territory was similar to diversity over the whole geographic range. This is best exemplified by the virus diversity observed in Switzerland or Czech Republic. These two countries yielded most of the known European subtype Eu3 subgroup sequences, and the diversity of viruses found within each of these small countries is comparable to that of the whole Eu3 subgroup, which is prevalent all over Central and Eastern Europe. Most of the deep tree nodes within all three established TBEV subtypes dated less than 300 years back. This could be explained by the recent emergence of most of the known TBEV diversity. Results of bioinformatics analysis presented here, together with multiple field findings, suggest that TBEV may be regarded as an emerging disease.

Emerging Concepts and Challenges in Rheumatoid Arthritis Gene Therapy.

Rheumatoid arthritis (RA) is a systemic inflammatory joint disease affecting about 1% of the population worldwide. Current treatment approaches do not ensure a cure for every patient. Moreover, classical regimens are based on nontargeted systemic immune suppression and have significant side effects. Biological treatment has advanced considerably but efficacy and specificity issues remain. Gene therapy is one of the potential future directions for RA therapy, which is rapidly developing. Several gene therapy trials done so far have been of moderate success, but experimental and genetics studies have yielded novel targets. As a result, the arsenal of gene therapy tools keeps growing. Currently, both viral and nonviral delivery systems are used for RA therapy. Herein, we review recent approaches for RA gene therapy.

The Effect of Sample Bias and Experimental Artefacts on the Statistical Phylogenetic Analysis of Picornaviruses.

Statistical phylogenetic methods are a powerful tool for inferring the evolutionary history of viruses through time and space. The selection of mathematical models and analysis parameters has a major impact on the outcome, and has been relatively well-described in the literature. The preparation of a sequence dataset is less formalized, but its impact can be even more profound. This article used simulated datasets of enterovirus sequences to evaluate the effect of sample bias on picornavirus phylogenetic studies. Possible approaches to the reduction of large datasets and their potential for introducing additional artefacts were demonstrated. The most consistent results were obtained using "smart sampling", which reduced sequence subsets from large studies more than those from smaller ones in order to preserve the rare sequences in a dataset. The effect of sequences with technical or annotation errors in the Bayesian framework was also analyzed. Sequences with about 0.5% sequencing errors or incorrect isolation dates altered by just 5 years could be detected by various approaches, but the efficiency of identification depended upon sequence position in a phylogenetic tree. Even a single erroneous sequence could profoundly destabilize the whole analysis by increasing the variance of the inferred evolutionary parameters.

Using Statistical Phylogenetics for Investigation of Enterovirus 71 Genotype A Reintroduction into Circulation.

Neurovirulent enterovirus 71 (EV-A71) caused a massive epidemic in China in 2008-2011. While subgenotype C4 was the major causative agent, a few isolates were almost identical to the prototype EV-A71 strain and belonged to genotype A. This variant was allegedly extinct since 1970, and its identification in this epidemic suggests reintroduction of the archive virus. Regression analysis of genetic distances (TempEst software) was of moderate utility due to the low resolution of classical phylogenetic methods. Bayesian phylogenetic analysis (BEAST software) suggested artificial introduction event based on highly aberrant phylogenetic tree branch rates that differed by over three standard deviations from the mean substitution rate for EV71. Manual nucleotide-level analysis was used to further explore the virus spread pattern after introduction into circulation. Upon reintroduction, the virus accumulated up to seven substitutions in VP1, most of them non-synonymous and located within the capsid's canyon or at its rims, compatible with readaptation of a lab strain to natural circulation.

Epigenetic Changes in the Pathogenesis of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects about 1% of the world's population. The etiology of RA remains unknown. It is considered to occur in the presence of genetic and environmental factors. An increasing body of evidence pinpoints that epigenetic modifications play an important role in the regulation of RA pathogenesis. Epigenetics causes heritable phenotype changes that are not determined by changes in the DNA sequence. The major epigenetic mechanisms include DNA methylation, histone proteins modifications and changes in gene expression caused by microRNAs and other non-coding RNAs. These modifications are reversible and could be modulated by diet, drugs, and other environmental factors. Specific changes in DNA methylation, histone modifications and abnormal expression of non-coding RNAs associated with RA have already been identified. This review focuses on the role of these multiple epigenetic factors in the pathogenesis and progression of the disease, not only in synovial fibroblasts, immune cells, but also in the peripheral blood of patients with RA, which clearly shows their high diagnostic potential and promising targets for therapy in the future.

Established and Emerging Strategies for Drug Delivery Across the Blood-Brain Barrier in Brain Cancer.

Brain tumors are characterized by very high mortality and, despite the continuous research on new pharmacological interventions, little therapeutic progress has been made. One of the main obstacles to improve current treatments is represented by the impermeability of the blood vessels residing within nervous tissue as well as of the new vascular net generating from the tumor, commonly referred to as blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB), respectively. In this review, we focused on established and emerging strategies to overcome the blood-brain barrier to increase drug delivery for brain cancer. To date, there are three broad strategies being investigated to cross the brain vascular wall and they are conceived to breach, bypass, and negotiate the access to the nervous tissue. In this paper, we summarized these approaches highlighting their working mechanism and their potential impact on the quality of life of the patients as well as their current status of development.