Evaluation of Hazara Orthonairovirus infection in tick cell lines as a model to study persistent infection of Crimean-Congo Hemorrhagic Fever Virus in ticks

Crimean-Congo hemorrhagic fever (CCHF) is a severe disease for humans caused by CCHF Orthonairovirus (CCHFV), a class 4 pathogen. Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector. CCHFV can be transmitted to its hosts during the tick blood feeding. It has been shown that CCHFV can persistently infects Hyalomma-derived tick cell lines without any cytopathic effect. However, the mechanism allowing the establishment of a persistent viral infection in ticks is still unknown. It has been recently reported that Hazara Orthonairovirus (HAZV) can be used as a BSL-2 viral model instead of CCHFV to study viral/vector interaction. The aim of our study is to elucidate the mechanism allowing the establishment of the CCHFV persistent infection in ticks by using HAZV as a model. We used classical and molecular methods applied to virology to characterize the establishment of the HAZV persistent infection in two Hyalomma anatolicum-derived cell lines, Hae/CTVM8 and Hae/CTVM9. As for CCHFV, we showed that HAZV persistently infects tick cells without any sign of cytopathic effect, and that cells can be cultured for more than two years. The persistent infection is established in roughly 10 days post-infection and viral titer is maintained at lower level in comparison to the earlier time points. Interestingly, short viral derived DNA forms (vDNAs) start to be detected in parallel with the beginning of viral replication and are maintained in persistently infected cells. Experiments with the antiretroviral drug AZT suggest that vDNAs are produced by a reverse transcriptase activity; furthermore, we collected evidence that vDNAs are not integrated and require an active viral genome replication. Morover, the presence of vDNA correlates with the downregulation of viral replication to promote viral tolerance and cell survival. In conclusion, vDNAs synthesis might represent a strategy to control viral replication in ticks, as recently demonstrated in insect, allowing the persistent infection in viral vectors