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Background  Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by  Culicoides  spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for  in vitro  virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines.    Methods  The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized  Culicoides sonorensis  midges was investigated. Virus strains were generated using reverse genetics and their growth was examined  in vitro . A laboratory colony of  C. sonorensis,  a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies.    Results  Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within  C. sonorensis  after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within  Culicoides .    Conclusion  The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.

Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis