Introduction. Bats are recognized as primary natural reservoirs for alpha- and betacoronaviruses. The interspecies transmission of bat coronaviruses to other mammalian hosts, including livestock and humans, can lead to epidemics, epizootics, and global pandemics. Objective. This study aims to describe coronaviruses associated with horseshoe bats (Rhinolophus spp.) in the southern regions of the European part of Russia. Materials and methods. Fecal samples were collected from bats inhabiting caves on the southern macroslope of the Greater Caucasus (Sochi-Adler region) during 2020, 2021, and 2024. Viral genomes were detected and analyzed using high-throughput sequencing (NGS) and RT-PCR. Results. A novel alphacoronavirus, designated Kudep virus (GenBank acc. # PQ649435), was identified in R. ferrumequinum. Presumably the Kudep virus represents a novel species within the subgenus Decacovirus of the genus Alphacoronavirus. The virus Showed 72% nucleotide identity to a Cardioderma bat coronavirus from Kenya and up to 67% nucleotide identity to the YN2012 virus group found in horseshoe bats in China. RT-PCR screening revealed active circulation of both Kudep virus and the previously described SARS-like betacoronavirus Khosta-1 in the study area. Infection rates in a single R. ferrumequinum colony during autumn 2021 reached 59.2% and 70.5% for Kudep and Khosta-1, respectively. Frequent co-infections with both viruses were observed in individual bats. Conclusion. Our findings expand the understanding of the distribution of bat alphacoronaviruses and their genetic diversity. We demonstrate the presence of a persistent natural foci of two potentially zoonotic bat coronaviruses, ecologically associated with R. ferrumequinum in the southern European part of Russia.

Introduction. There are natural foci of Crimean-Congo hemorrhagic fever (CCHF) that vectored by Hyalomma marginatum ticks in Volga river delta (Astrakhan region, South of Russia). The circulation of Dhori virus (DHOV) (Thogotovirus: Orthomyxoviridae) has been also shown here. We hypothesized that other tick-borne arboviruses are also likely to circulate in the region. In particular, Bhanja virus (Phlebovirus: Phenuiviridae), Wad Medani virus (Orbivirus: Reoviridae), and Tamdy virus (Orthonairovirus: Nairoviridae), which were found to circulate in neighboring regions and are vectored by Haemaphysalis spp., Dermacenter spp., and Hyalomma spp. ticks. Goals and objectives. The aim of the study was to examine ixodid ticks in Volga river delta for the presence of CCHFV, DHOV, Bhanja virus, Wad Medani virus, and Tamdy virus. Material and methods. Ticks were collected in Volga river delta in 2017. We used molecular genetic methods for the detection and analysis of nucleic acids (PCR, sequencing, phylogenetic analysis). Results. We detect CCHFV and DHOV RNA in H. marginatum ticks. The rate of infected H. marginatum ticks was 1.98% for CCHFV and 0.4% for DHOV. The results of genetic analysis showed that found DHOV strains are almost identical (99-100% in the M gene) and forms a separate genetic lineage alongside of Batken virus from Central Asia. At the same time, Bhanja virus, Wad Medani virus, and Tamdy virus were not found in ticks, collected in this region. Conclusions. DHOV is circulating in the natural foci of CCHF in the Volga river delta. The ratio of infection of H. marginatum with CCHFV and DHOV was determined for the first time.

Chenuda virus (CNUV) (Orbivirus, Reoviridae) is the only known orbivirus associated with argas (Argasidae) ticks. Scientific study of this group is necessary for understanding of Orbivirus genus evolution patterns. We conducted a comparative analysis of full genomes of five different viruses of Chenuda virus species, including Baku virus strains (BAKV) circulating in a rather limited area in the Central Asia and Transcaucasia. It was shown that VP4(OC1) and VP6(Hel) proteins variability greatly exceeds the variability of other proteins. The divergence between CNUV and BAKV in this proteins is about 50%. Even in closely related strains isolated from the same geographical region, the conservative genes of which are 90-95% identical, the VP4(OC1) and VP6(Hel) divergence reaches values that would usually be indicative of different serotypes (74.1-82.2%).

Chenuda virus (CNUV) (Orbivirus, Reoviridae) is the only known orbivirus associated with argas (Argasidae) ticks. Scientific study of this group is necessary for understanding of Orbivirus genus evolution patterns. We conducted a comparative analysis of full genomes of five different viruses of Chenuda virus species, including Baku virus strains (BAKV) circulating in a rather limited area in the Central Asia and Transcaucasia. It was shown that VP4(OC1) and VP6(Hel) proteins variability greatly exceeds the variability of other proteins. The divergence between CNUV and BAKV in this proteins is about 50%. Even in closely related strains isolated from the same geographical region, the conservative genes of which are 90-95% identical, the VP4(OC1) and VP6(Hel) divergence reaches values that would usually be indicative of different serotypes (74.1-82.2%).

Almost complete nucleotide sequences for the S, M, and L segments were obtained for three strains of the Batai virus (Bunyamwera serogroup, genus Orthobunyavirus, Bunyaviridae family). Based on the results of the phylogenetic analysis conducted forthe three genomic segments LEIV Ast507 and LEIV-Ast528 strains were grouped with other European BATV isolates and were found to be almost identical to the strain 42 isolated from Volgograd Region, Russia, 2003. Surprisingly, LEIV-13395 strain isolated from the Aedes sp. mosquitos in Magadan Oblast, 1987, turned out to be a novel genotype inside Bunyamwera serogroup. The highest nucleotide identity levels of LEIV-13395 genomicsegments (86.9%, 80.8%, 79.7% for S, M and L segments respectively) were observed with corresponding segments of the Batai virus.

Unidentified Khurdun virus (KHURV) was isolated in 2001 from coot (Fulica atra, linnaeus, 1758) in the Volga River delta (Astrakhan Region, Russian Federation). Here we report that the KHUV genome was de novo sequenced (on illumina platform) and the KHURV was classified as a novel prototypic bunyavirus. The KHURV genome comprises three negative-sense RNA segments (L, M, and s); its terminal nucleotide sequences are canonical for the Orthobunyavirus genus. Based on the results of the molecular-genetic and phylogenetic analysis we suggest that the KHuRv belongs to the genus Orthobunyavirus (Bunyaviridae).

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes. Out of 37 isolates, 27 were reassortants. The majority of the reassortants contained the HA gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus. The data demonstrate the previously unrecognized phenomenon of segment-specific deviation from the random distribution of parent genes in the reassortants. The results are discussed in connection with the problem of differential competition between influenza A virus gene segments in mixed infection and random versus non-random reassortment of gene segments under non-selective conditions.