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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » ABADRU » Research » Publications at this Location » Publication #321596
Title: Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

Author
item FEENSTRA, FEMKE - Central Veterinary Institute
item Drolet, Barbara
item BOONSTRA, J - Central Veterinary Institute
item VAN RIJN, PIET - Central Veterinary Institute

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2015
Publication Date: 9/17/2015
Publication URL: http://handle.nal.usda.gov/10113/62676
Citation: Feenstra, F., Drolet, B.S., Boonstra, J., Van Rijn, P.A. 2015. Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis. Parasites & Vectors. 8:476-484. doi:10.1186/s13071-015-1063-3.

Interpretive Summary: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. After deleting viral genetic information that make proteins called NS3/NS3a the virus was able to multiply but was delayed in escaping from cells. When the virus was put in sheep, not only did the mutant not cause disease, it protected the sheep from infection with the wildtype virus. Therefore it is considered to be a 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. We investigated the ability of this vaccine candidate to infect and disseminate within the insect vector, Culicoides sonorensis. The vaccine candidate virus was unable to infect the insect. This demonstrates that NS3/NS3a is required for viral replication within Culicoides and clearly demonstrates the inability of NS3/NS3a knockout vaccine strains to be transmitted by midges.

Technical Abstract: 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.