KINETICS OF HAZARA VIRUS REPLICATION IN ARTIFICIALLY INFECTED TICKS

Crimean-Congo hemorrhagic fever (CCHF) is the most important Tick-borne viral disease of humans, and it is caused by a negative-sense single-stranded RNA virus with the homonymous name (CCHFV). CCHF is mainly transmitted through the bite of Hyalomma ticks but also Ixodes ricinus, that is widely distributed in Europe, seems to represent a potential vector for the pathogen. Very little is known about the factors involved in the vector-pathogen interaction and on the persistence of CCHFV in ticks due to the requirement for the virus to be handled in biosafety level (BSL)-4 facilities. The aim of this research project is the optimization of an artificial infection protocol to study the vector competence of I. ricinus, employing Hazara virus (HAZV) as a BSL-2 surrogate model for CCHFV. The immersion method was utilized to infect larvae, nymphs, and adults of I. ricinus with different concentrations of HAZV (2,5x10^3 and 6,4x10^6 PFU/ml). Ticks were sacrificed at 3- and 7- days post infection (d.p.i.) to detect and quantify HAZV in homogenates of whole ticks or in specific organs (salivary glands and midgut) by qRT-PCR. Larvae were analysed in pools of three, while nymphs and adults were individually tested for HAZV infection. HAZV RNA was detected in almost 100% of ticks using the higher concentration of the virus while a few ticks resulted positive at the lower HAZV concentration. No increasing in the amount of virus was noticed in larvae 3- and 7-days post infection. Instead, HAZV increased from 10^4 to 10^5 PFU/ml in nymphs. The same variation was also highlighted in total homogenates of adult females. In the salivary glands of adult females and males, the virus load increased as well, from 10^3 to 10^4 PFU/ml. An order of magnitude difference was also found 7 d.p.i. compared to 3 d.p.i. in the midgut of adult males (from 10^5 to 10^6 PFU/ml). The presence of viral particles in these organs is an important information to study the vector competence of I. ricinus ticks for HAZV. No infection-associated tick mortality was observed. Nevertheless, it will be necessary to infect more samples and to increase the vitality of ticks to analyse the trend of the viral replication in longest time points.