ADAPTATIVE EVOLUTION OF HAZARA ORTHONAIROVIRUS DURING INFECTION OF HYALOMMA-DERIVED TICK CELLS

Aim of the Study: Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne pathogen that presents a serious threat to human health, causing a severe hemorrhagic fever with high mortality. Ticks of the genus Hyalomma are the main vectors and reservoirs, harboring the virus persistently. However, virus-vector interactions remain poorly understood due to biosafety constraints. Host cycling likely contributes to viral flexibility, however cross-species transmission imposes selective pressures shaping adaptation. This study investigated the evolution of Hazara virus (HAZV), a model for CCHFV, across host cell lines to elucidate how host-specific pressure drives viral adaptation and infectivity. Methods Used: HAZV grown in SW13 cells was used to infect the Hyalomma tick cell line HAE/CTVM8. Samples were collected at 30, 60 and 90 days, followed by four additional passages each lasting 30 days. Genotypic changes were assessed by NGS on cell pellets and supernatants. Wild-type and tickadapted viruses were compared in mammalian cell lines (SW-13, Vero CCL-81, Huh7, and A549). Viral replication was measured by RT-qPCR, nucleoprotein (NP) expression by immunofluorescence (IF), and viral spread by plaque assay. Results and Conclusions: HAZV propagation in HAE/CTVM8 cells led to mutations within all genome segments, with stabilization of mutations in the polymerase and glycoprotein precursor genes. Tick-adapted viruses showed higher intracellular RNA but lower titers in the supernatant, suggesting impaired release. NP displayed a diffuse intracellular distribution compared to the ER–Golgi-associated pattern in WT infection. Apoptosis induction was time- and cell line-dependent: in Huh7 cells levels were similar to WT, A549 cells showed increased early and late apoptosis, and in SW13 cells viability decreased at 48-72hpi. In A549 cells, only WT virus formed plaques, while adapted viruses caused diffuse cell death suggesting increased interferon (IFN) sensitivity. Treatment with Ruxolitinib restored plaque formation, confirming IFN-mediated restriction. Collectively, HAZV adaptation in tick cells results in a trade-off phenotype in mammalian cells, with efficient intracellular replication, increased apoptosis, and greater IFN sensitivity limiting viral spread.