In this work, we presented data from a two-year study of flavi-, flavi-like, and phenuiviruses circulation in the population of ixodid ticks in the Chelyabinsk region. We isolated three tick-borne encephalitis virus (TBEV) strains from I. persulcatus, which was not detected in the ticks of the genus Dermacentor. The virus prevalence ranged from 0.66% to 2.28%. The Yanggou tick virus (YGTV) is widespread in steppe and forest-steppe zones and is mainly associated with ticks of the genus Dermacentor. We isolated 26 strains from D. reticulatus, D. marginatus, and I. persulcatus ticks in the HAE/CTVM8 tick cell line. The virus prevalence ranged from 1.58% to 4.18% in D. reticulatus, ranged from 0.78% to 3.93% in D. marginatus, and was 0.66% in I. persulcatus. There was combined focus of TBEV and YGTV in the territory of the Chelyabinsk region. The Alongshan virus (ALSV) was found to be associated with I. persulcatus ticks and is spread in forest zone. We detected 12 amplicons and isolated 7 strains of ALSV in tick cells. The virus prevalence ranged from 1.13% to 6.00%. The phlebovirus Gomselga and unclassified phenuivirus Stavropol were associated with I. persulcatus and D. reticulatus ticks, respectively. Virus prevalence of the unclassified phenuivirus Stavropol in the Chelyabinsk region is lower than that in neighbouring regions.

Many viruses are known to trigger endoplasmic reticulum (ER) stress in host cells, which in turn can develop a protective unfolded protein response (UPR). Depending on the conditions, the UPR may lead to either cell survival or programmed cell death. One of three UPR branches involves the upregulation of Xbp1 transcription factor caused by the unconventional cytoplasmic splicing of its mRNA. This process is accomplished by the phosphorylated form of the endoribonuclease/protein kinase Ire1/ERN1. Here, we show that the phosphorylation of Ire1 is up-regulated in HeLa cells early in enterovirus infection but down-regulated at later stages. We also find that Ire1 is cleaved in poliovirus- and coxsackievirus-infected HeLa cells 4–6 h after infection. We further show that the Ire1-mediated Xbp1 mRNA splicing is repressed in infected cells in a time-dependent manner. Thus, our results demonstrate the ability of enteroviruses to actively modulate the Ire1-Xbp1 host defensive pathway by inducing phosphorylation and proteolytic cleavage of the ER stress sensor Ire1, as well as down-regulating its splicing activity. Inactivation of Ire1 could be a novel mode of the UPR manipulation employed by viruses to modify the ER stress response in the infected cells.

The tick-borne encephalitis virus (TBEV) is one of the most common members of the Orthoflavivirus genus, which comprises the causative agents of severe diseases in humans and animals. Due to the expanding areas of orthoflavivirus infection, its differential diagnosis is highly demanded. Commercial test kits based on inactivated TBEV may not provide reliable differentiation between flaviviruses because of serological crossover in this genus. Application of recombinant domains (sE and dIII) of the TBEV Sukhar-strain protein E as antigens in an ELISA test system allowed us to identify a wide range of antibodies specific to different TBEV strains. We tested 53 sera from human patients with confirmed TBE diagnosis (the efficacy of our test system based on sE protein was 98%) and 56 sera from patients with other orthoflavivirus infections in which no positive ones were detected using our ELISA test system, thus being indicative of its 100% specificity. We also tested mouse and rabbit sera containing antibodies specific to 17 TBEV strains belonging to different subtypes; this assay exhibited high efficacy and differentiation ability in detecting antibodies against TBEV from other orthoflaviviruses such as Omsk hemorrhagic fever, Powassan, yellow fever, dengue, West Nile, Zika, and Japanese encephalitis viruses.

The family Phenuiviridae, part of the order Hareavirales, includes arboviruses and arthropod-associated viruses, with sandflies, mosquitoes, and ticks as primary vectors. Historically, only sandfly/mosquito-borne phenuiviruses were associated with human diseases, but the emergence of severe fever with thrombocytopenia syndrome (SFTS) has highlighted the potential of tick-borne phenuiviruses as human pathogens. Recent discoveries of new arthropod-associated viruses, some of which remain unclassified, underscore the need for sensitive detection and differentiation methods, particularly in regions where these viruses may co-circulate. This study aimed to develop real-time PCR test systems for identifying and differentiating unclassified viruses within the Phenuiviridae family. In this study, tick suspensions containing phenuiviruses, previously obtained during the screening of ticks from various regions of Russia using pan-phenuivirus primers, were used. Specific primers and probes were designed to differentiate five Phenuiviridae viruses of genera Uukuvirus, Ixovirus, Phlebovirus and one unclassified phenuivirus, and their analytical sensitivity and specificity were evaluated. These PCR-based tools provide a robust method for detecting and classifying uncharacterized phenuiviruses, contributing to improved surveillance and understanding their potential epidemiological and epizootological impacts.

BACKGROUND: The emergence of new variants of the SARS-CoV-2 virus at the end of 2020, which became a source of increased risk for global public health, prompted the study of their molecular biological characteristics and pathogenic effects. It is known that one of the reasons for the viruses pathogenic action is their interaction with the defense mechanisms of the cell. Ire1 (inositol-requiring enzyme 1)-mediated splicing of Xbp1 mRNA (X-box binding protein 1) is a protective mechanism that is activated in response to the accumulation of misfolded proteins in the cell, a situation that occurs due to uncontrolled synthesis of viral proteins during infection. Studying the interaction of different variants of the SARS-CoV-2 virus with this protective mechanism will help to shed light on various aspects of the pathogenesis of a new coronavirus infection. AIM: Study of Ire1-Xbp1 defense mechanism activation and modulation in SARS-CoV-2 infected Vero cells. MATERIALS AND METHODS: We studied the activation of the Ire1 enzyme in Vero cells infected with various variants of the SARS-CoV-2 virus using immunoblotting and antibodies to various forms of this protein in the cell. In addition, we studied the activation of Xbp1 mRNA splicing under conditions of infection with various variants of the SARS-CoV-2 virus in a PCR reaction with specific primers. RESULTS: Reproduction of B.1.1.529 (Omicron) strain in Vero cells is slower than B.1.1.7 (Alpha) and B.1.617.2 (Delta) strains of SARS-CoV-2. The whole reproduction cycle is 48 hours. Ire1-dependent defense mechanism is activated after 12 hours of SARS-CoV-2 infection with either of three variants. However, despite the activation of the Ire1 endonuclease domain, there is no Xbp1 mRNA splicing in SARS-CoV-2 infected cells. Inhibition of Xbp1 mRNA splicing occurs slower in Vero cells infected with the B.1.1.529 Omicron variant. CONCLUSIONS: The paper describes the reproduction of various variants of the SARS-CoV-2 virus in Vero cell culture and the activation of the Ire1-Xbp1 defense mechanism during infection. The Ire1 endonuclease is phosphorylated, however, mRNA splicing of the Xbp1 transcription factor is impaired in SARS-CoV-2 infected cells. A decrease in the rate of inhibition of this protective mechanism in Vero cells infected with the Omicron (B.1.1.529) variant of the SARS-CoV-2 virus may be the reason for its lower pathogenicity described in various studies.