EVALUATION OF HAZARA VIRUS INFECTION IN ARTIFICIALLY INFECTED TICKS

Crimean-Congo hemorrhagic fever (CCHF) is a widespread tick-borne disease caused by CCHF virus (CCHFV), a member of the Nairoviridae family. CCHFV is mainly transmitted through the bite of Hyalomma ticks, whose climate change-driven spread into new areas represents a serious threat to public health. CCHFV has also been detected in Ixodes ricinus, the most abundant tick species in Europe. However, its role as a vector has not yet been demonstrated. The factors involved in the vector-virus interaction are still poorly characterized, due to the requirement for the virus to be handled in BSL-4 facilities. The aim of this study is to evaluate the vector competence of I. ricinus for CCHFV using Hazara virus (HAZV), a non-pathogenic nairovirus used as a model for CCHFV. Since the infection of ticks through feeding is complex due to the need for viremic animal hosts, the immersion method was employed. This protocol could be easier than the animal model for infecting ticks in high biocontainment conditions [1]. Larvae, nymphs, and adults of I. ricinus were infected with 10^8 and 10^11 FFU/mL of HAZV. Ticks were sacrificed at 3- and 7- days post infection (d.p.i.) to detect and quantify HAZV by qRT-PCR. Preliminary results showed almost 100% of the ticks were infected with HAZV. HAZV RNA copies increased in larvae from 3.00 x 10^3 FFU/mL to 6.33 x 10^3 FFU/mL and from 6.23 x 10^3 FFU/mL to 9.31 x 10^3 FFU/mL between 3 and 7 d.p.i., using 10^8 and 10^11 FFU/mL HAZV concentrations, respectively. A similar result was observed in nymphs infected with 10^11 FFU/mL HAZV (from 9.27 x 10^3 FFU/mL to 1.74 x 10^4 FFU/mL), and in adult males infected with 10^8 FFU/mL HAZV (from 1.16 x 10^4 FFU/mL to 2.36 x 10^4 FFU/mL). HAZV RNA copies remained almost unchanged in nymphs infected with HAZV at lower concentration and in adults infected with 10^11 FFU/mL HAZV. The analyses of the other experimental replicates are still ongoing and will be compared with those obtained from Hyalomma marginatum ticks, a primary vector of CCHFV used as a control. These data suggest potential viral replication in some of the tick life stages; however, longer time-points are required to better characterized viral replication. Detection of viral DNA forms will be assessed as an additional marker of viral replication in ticks. Analysis of salivary glands to localize HAZV plays a key role in evaluating the potential transmissibility of the virus and will also be performed.