The Persistent Infection of Tick Cells by Hazara orthonairovirus Is Mediated by Virus-Derived DNA Forms

Introduction: Crimean–Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF orthonairovirus (CCHFV), a class 4 pathogen. <i>Hyalomma </i>ticks are the viral reservoir, and they represent the main vector. CCHFV can be transmitted to its hosts during tick blood feeding. We have previously shown that CCHFV can persistently infect <i>Hyalomma</i>-derived tick cell lines without any cytopathic effect. However, the mechanism allowing for 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 model virus instead of CCHFV to study viral/vector interaction. The aim of our study is to elucidate the mechanism that allows establishment of persistent CCHFV infection in ticks using HAZV as a model. Methods: We used classical and molecular methods applied to virology to characterize the establishment of persistent HAZV infection in two <i>Hyalomma</i><i> </i><i>anatolicum</i>-derived cell lines—HAE/CTVM8 and HAE/CTVM9. Results: As for CCHFV, we showed that HAZV persistently infects tick cells without any sign of cytopathic effect and that infected cells can be cultured for more than one year. The persistent infection is characterized by a low viral titer compared to the initial 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 retrotranscriptase activity. Furthermore, we collected evidence that vDNAs are not integrated and seem to be involved in the downregulation of viral replication by promoting cell survival. Conclusion: vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks, as recently demonstrated in insects, allowing for persistent infection of virus vectors.