Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

Subgenomic Flaviviral RNAs of Dengue Viruses.

Subgenomic flaviviral RNAs (sfRNAs) are produced during flavivirus infections in both arthropod and vertebrate cells. They are undegraded products originating from the viral 3' untranslated region (3' UTR), a result of the action of the host 5'-3' exoribonuclease, Xrn1, when it encounters specific RNA structures known as Xrn1-resistant RNAs (xrRNAs) within the viral 3' UTR. Dengue viruses generate three to four distinct species of sfRNAs through the presence of two xrRNAs and two dumbbell structures (DBs). The tertiary structures of xrRNAs have been characterized to form a ringlike structure around the 5' end of the viral RNA, effectively inhibiting the activity of Xrn1. The most important role of DENV sfRNAs is to inhibit host antiviral responses by interacting with viral and host proteins, thereby influencing viral pathogenicity, replicative fitness, epidemiological fitness, and transmission. In this review, we aimed to summarize the biogenesis, structures, and functions of DENV sfRNAs, exploring their implications for viral interference.