Sheep and Cattle Are Not Susceptible to Experimental Inoculation with Hazara Orthonairovirus, a Tick-Borne Arbovirus Closely Related to CCHFV

Abstract

Hazara orthonairovirus (HAZV) is a tick-borne arbovirus closely related to Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). Whereas CCHFV is a biosafety level (BSL) 4 agent, HAZV is classified as BSL 2, as it is not known to cause any disease in humans. Belonging to the same serogroup as CCHFV, HAZV might act as a model which can provide a better understanding of this important zoonosis. Furthermore, the serological relatedness may cause diagnostic problems if antibodies against HAZV interfere with current CCHFV serological assays. Therefore, sheep and cattle-important natural hosts for CCHFV-were experimentally infected with HAZV to prove their susceptibility and evaluate potential antibody cross-reactivities. According to this study, neither sheep nor cattle are susceptible to experimental HAZV infections. Consequently, the HAZV infection in ruminants is clearly distinct from CCHFV infections. Sera of immunized animals weakly cross-reacted between HAZV and CCHFV in immunofluorescence and immunoblot assays, but not in commercial CCHFV ELISAs commonly used for field studies.

Keywords: CCHFV; Crimean–Congo hemorrhagic fever; HAZV; Hazara orthonairovirus; animal model; cross-reactivity.

Figure 1

Rectal body temperature during the observation period: No fever could be detected in inoculated cattle (A) or sheep (B). Each animal is represented by a single pattern. Physiological rectal body temperatures range up to 39.5 °C for the calves and up to 40.0 °C for the sheep, respectively (illustrated by the red line in each graph); short term higher temperatures may be animal handling/excitement artefacts.

Figure 2

Indirect immunofluorescence assay with HAZV infected Vero E6 cells: serum prior immunization did not lead to positive results in non-infected controls (A) or infected cells (B). Post-immunization serum (1/50 diluted) lead to a specific fluorescence signal in infected cells (D) whereas non-infected controls (C) did not show specific staining patterns and are therefore evaluated as negative. Panel I shows the immunized calf (HAZV-IMMU-calf-1) and Panel II the immunized sheep (HAZV-IMMU-sheep-1) results.

Figure 3

Indirect bovine HAZV N protein ELISA: corrected OD values for the 100 negative reference sera from German cattle demonstrate 98% specificity of this ELISA. OD values were below the determined cut-off for D5, D6, E5 and F2 prior and post-inoculation. Calf E6 tested positive prior inoculation.

Figure 4

Indirect ovine HAZV N protein ELISA: corrected OD values for the 100 negative reference sera from German sheep demonstrate 98% specificity of this ELISA. OD values were below the determined cut-off for all HAZV inoculated sheep.

Figure 5

Indirect immunofluorescence assay with HAZV infected Vero E6 cells: no differences between wells with serum prior infection ((A): non-infected controls, (B): infected wells) and wells with serum 28 dpi ((C): non-infected controls, (D): infected wells) were registered in infected calves ((I), example calf E5)) and sheep ((II), example sheep A5)).

Figure 6

Western blot analysis with recombinant HAZV (H) and CCHFV (C) N protein: HAZV antiserum reacted strongly with HAZV N protein and showed weak cross-reaction with CCHFV N protein when tested with calf (HAZV-IMMU-calf-1, panel (A)) and sheep (HAZV-IMMU-sheep-1, panel (B)) post-immunization sera (E). Pre-immunization sera (P) did not lead to positive results.

Figure 7

Indirect immunofluorescence assay (Euroimmun, Luebeck) A+D (cell control), B+E (CCHFV GPC, glycoprotein C), C+F (CCHFV N): Sera prior immunization (A–C) and post-immunization (D–F) are depicted for the immunized calf (HAZV-IMMU-calf-1, panel I) and sheep (HAZV-IMMU-sheep-1, panel II). A specific fluorescence signal could be detected for both post-immunization sera on cells transfected with CCHFV GPC and CCHFV N, whereas sera prior immunization tested negative.