Cu(II)-Based Paramagnetic Probe to Study RNA-Protein Interactions by NMR.

Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.

A highly pathogenic avian influenza virus H5N1 clade 2.3.4.4 detected in Samara Oblast, Russian Federation.

Avian influenza (AI) is a global problem impacting birds and mammals, causing economic losses in commercial poultry farms and backyard settings. In 2022, over 8,500 AI cases were reported worldwide, with the H5 subtype being responsible for many outbreaks in wild and domestic birds. In the territory of the Russian Federation, outbreaks of AI have been massively reported since 2020, both among domestic bird species and wild bird species. Wild migratory birds often serve as natural reservoirs for AI viruses, and interactions between bird species can lead to the emergence of new, highly pathogenic variants through genetic recombination between strains. In order to combat the widespread outbreaks of the disease and potential risks of further spread in 2021, monitoring studies were conducted in the Samara Oblast, the southeastern region of European Russian Federation. These studies aimed to diagnose and characterize circulating AI virus variants among wild migratory birds during waterfowl hunting in areas of mass nesting. Among the 98 shot birds, a highly pathogenic A/H5N1 AI virus was detected in a Eurasian Teal from the Bolshechernigovsky district. It was classified into clade 2.3.4.4 based on the cleavage site structure of HA. Phylogenetic analysis showed a high relatedness of the identified strain in the Samara Oblast with field isolates from Russia, Nigeria, Bangladesh, and Benin. The article emphasizes the importance of monitoring AI virus spread in both wild and poultry, highlighting the need for timely information exchange to assess risks. Further comprehensive studies are necessary to understand virus dissemination pathways.

Risk Factors of African Swine Fever in Domestic Pigs of the Samara Region, Russian Federation.

African swine fever (ASF) is an incurable viral disease of domestic and wild pigs. A large-scale spread of ASF began in Eurasia in 2007 and has affected territories from Belgium to the Far East, occurring as both local- and regional-level epidemics. In 2020, a massive ASF epidemic emerged in the southeastern region of European Russia in the Samara Oblast and included 41 outbreaks of ASF in domestic pigs and 40 cases in wild boar. The Samara Oblast is characterized by a relatively low density of wild boar (0.04-0.05 head/km2) and domestic pigs (1.1-1.3 head/km2), with a high prevalence of small-scale productions (household farms). This study aims to understand the driving forces of the disease and perform a risk assessment for this region using complex epidemiological analyses. The socioeconomic and environmental factors of the ASF outbreak were explored using Generalized Linear Logistic Regression, where ASF infection status of the Samara Oblast districts was treated as a response variable. Presence of the virus in a district was found to be most significantly (p < 0.05) associated with the importation of live pigs from ASF-affected regions of Russia (OR = 371.52; 95% CI: 1.58-87290.57), less significantly (p < 0.1) associated with the density of smallholder farms (OR = 2.94; 0.82-10.59), volume of pork products' importation from ASF-affected regions of Russia (OR = 1.01; 1.00-1.02), summary pig population (OR = 1.01; 0.99-1.02), and insignificantly (p > 0.1) associated with presence of a common border with an ASF-affected region (OR = 89.2; 0.07-11208.64). No associations were found with the densities of pig and wild boar populations. The colocation analysis revealed no significant concentration of outbreaks in domestic pigs near cases in wild boar or vice versa. These results suggest that outbreaks notified in low biosecurity household farms were mainly associated with the transportation and trade of pigs and pork products from ASF-affected regions of Russia. The findings underline the importance of taking into account animal transportation data while conducting future studies to develop a risk map for the region and the rest of European Russia.

Cobalt-based paramagnetic probe to study RNA-protein interactions by NMR.

Paramagnetic resonance enhancement (PRE) is an NMR technique that allows studying three-dimensional structures of RNA-protein complexes in solution. RNA strands are typically spin labeled using nitroxide reagents, which provide minimal perturbation to the native structure. The current work describes an alternative approach, which is based on a Co2+-based probe that can be covalently attached to RNA in the vicinity of the protein's binding site using 'click' chemistry. Similar to nitroxide spin labels, the transition metal based probe is capable of attenuating NMR signal intensities from protein residues localized <40Å away. The extent of attenuation is related to the probe's distance, thus allowing for construction of the protein's contact surface map. This new paradigm has been applied to study binding of HIV-1 nucleocapsid protein 7, NCp7, to a model RNA pentanucleotide.