Data from studies with CCHFV L protein showed that the OTU domain is enough to inhibit total protein ubiquitination and ISG15ylation in 293T cells [24] . We wanted to know if NSDV/GV has any effects on total protein ubiquitination and/ or ISG15ylation in cells. We investigated this using Vero cells transfected with expression constructs for tagged forms of Ub or ISG15, with appropriate supporting plasmids as required for the ISG15 system [24, 39] . Cells were transfected with a plasmid expressing HA-tagged ubiquitin and subsequently infected with the GV or NSDV isolates. Twelve hours post-infection cell extracts were prepared and the expression of ubiquitin conjugated-proteins determined by immunoblotting ( fig. 7a) , showing that infection with either isolate caused a decrease in total protein ubiquitination compared to uninfected cells. In a similar way we studied the effects of NSDV/GV on ISG15ylation of proteins in infected host cells. ISG15 is a Ubl that is very rapidly induced in type I IFN-treated cells [40] and is thought to play important roles in anti-viral responses, either as a monomer or by conjugation to host cell proteins (reviewed in [41] ). ISG15 conjugation to host cell proteins exerts antiviral activity against influenza virus [42] and Sindbis virus [43] . For our studies on NSDV and its effects on host ISGylation during infection we made use of the fact that ISG15ylation can also be generated by transfecting expression constructs for ISG15 along with plasmids encoding the core components of the ISG15 conjugation system, the E1 activating protein (mUBE1L), E2 conjugating protein (UbcM8) and E3 ligase (mHerc6) into cells in the absence of IFN [39] . After transfecting these four plasmids into Vero cells, the cells were subsequently infected with the GV or NSDV isolates or left uninfected. Twelve hours post-infection cells were lysed and the level of ISG15-conjugates were examined by immunoblotting ( fig. 7b) . Infection of NSDV/ GV resulted in a drastic decrease in ISG15-conjugates found in Vero cells when compared to uninfected cells. Importantly, the infection with NSDV itself did not cause a reduction in the total amount of mono-ISG15 expressed from the transfected plasmid, as shown by tracks 5-7 of fig. 7b , where the helper plasmids were omitted, so one is simply comparing ISG15 expression in uninfected and infected cells. This excludes any indirect effect of infection on ISGylation through an effect on ISG15 expression or stability.