Novel segmented tick-borne RNA viruses belonging to the group of Jingmenviruses (JMVs) are widespread across Africa, Asia, Europe, and America. In this work, we obtained whole-genome sequences of two Kindia tick virus (KITV) isolates and performed modeling and the functional annotation of the secondary structure of 5′ and 3′ UTRs from JMV and KITV viruses. UTRs of various KITV segments are characterized by the following points: (1) the polyadenylated 3′ UTR; (2) 5′ DAR and 3′ DAR motifs; (3) a highly conserved 5′-CACAG-3′ pentanucleotide; (4) a binding site of the La protein; (5) multiple UAG sites providing interactions with the MSI1 protein; (6) three homologous sequences in the 5′ UTR and 3′ UTR of segment 2; (7) the segment 2 3′ UTR of a KITV/2017/1 isolate, which comprises two consecutive 40 nucleotide repeats forming a Y-3 structure; (8) a 35-nucleotide deletion in the second repeat of the segment 2 3′ UTR of KITV/2018/1 and KITV/2018/2 isolates, leading to a modification of the Y-3 structure; (9) two pseudoknots in the segment 2 3′ UTR; (10) the 5′ UTR and 3′ UTR being represented by patterns of conserved motifs; (11) the 5′-CAAGUG-3′ sequence occurring in early UTR hairpins. Thus, we identified regulatory elements in the UTRs of KITV, which are characteristic of orthoflaviviruses. This suggests that they hold functional significance for the replication of JMVs and the evolutionary similarity between orthoflaviviruses and segmented flavi-like viruses.

A large number of novel viruses potentially pathogenic for humans are discovered currently. Studying many of them by classical methods of virology is difficult due to the absence of a live viral particles or a sufficient amount of its genetic material. In this case, the use of modern methods of bioinformatics, synthetic and structural biology can help. Haseki tick virus (HSTV) is a recently discovered tick-borne unclassified ssRNA(+) virus. HSTV positive patients experienced fever and elevated temperature. However, at the moment, there is no information on the tertiary structure and functions of its proteins. In this work, we used AlphaFold 3 for annotation of HSTV protein functions, based on the principle that the tertiary structure of a protein is inextricably linked with its molecular function. We were the first to obtain models of tertiary structures and describe the putative functions of HSTV nonstructural proteins (NS3 helicase, NS3 protease, NS5 RNA-dependent RNA-polymerase, and NS5 methyltransferase). These proteins play a key role in the replication of the viral genome and can be targets for the development of direct-acting antiviral drugs.

Currently, a large number of novel tick-borne viruses potentially pathogenic to humans are discovered. Studying many of them by classical methods of virology is difficult due to the absence of live viral particles or a sufficient amount of their genetic material. In this case, the use of modern methods of bioinformatics and synthetic and structural biology can help. Haseki tick virus (HSTV) is a recently discovered tick-borne unclassified ssRNA(+) virus. HSTV-positive patients experienced fever and an elevated temperature. However, at the moment, there is no information on the tertiary structure and functions of its proteins. In this work, we used AlphaFold 3 and other bioinformatic tools for the annotation of HSTV nonstructural proteins, based on the principle that the tertiary structure of a protein is inextricably linked with its molecular function. We were the first to obtain models of tertiary structures and describe the putative functions of HSTV nonstructural proteins (NS3 helicase, NS3 protease, NS5 RNA-dependent RNA-polymerase, and NS5 methyltransferase), which play a key role in viral genome replication. Our results may help in further taxonomic identification of HSTV and the development of direct-acting antiviral drugs, POC tests, and vaccines.