Generation of a recombinant Bunyamwera virus expressing green fluorescent protein and its use in functional analysis of the viral nucleoprotein

Bunyamwera orthobunyavirus (BUNV) is a negative-sense, single-stranded RNA virus classified within the Orthobunyavirus genus of the Bunyavirales order; the prototype of both the genus and the order. BUNV is widely distributed throughout Africa, requiring arthropod vectors to transmit infection across a vast array of animal hosts and humans. Although BUNV infection in humans results in mild febrile illness, other members of the genus, such as Oropouche virus (OROV), La Crosse encephalitis virus (LACV), and Schmallenberg virus (SBV), are important global human or animal pathogens with much more severe indications. The ability for Orthobunyaviruses to reassort during co-infection, in combination with their worldwide distribution, poses a potential threat to human public health, animal health, and food security.
As a biosafety level II virus, BUNV can be used as an efficient and safer model system for studying the biology of other members of the Orthobunyavirus genus as it is relatively well understood relative to other members of the Peribunyaviridae family.
We have developed a novel BUNV reverse genetics rescue system using an eGFP reporter gene in the S segment, downstream of the nucleoprotein (N) gene, and a self-cleaving peptide sequence (P2A), to facilitate independent expression of eGFP and viral genes. This tool enables the visualisation of BUNV infection and measurement of BUNV gene expression in real-time.
This eGFP expressing recombinant virus was used to examine the importance of eight N protein residues suggested to have roles in genome assembly. Single amino acid substitutions were also generated in the S segment and attempts were made to rescue mutant viruses each bearing one of these residues changed to alanine. These point mutations permit the investigation of the formation of a correctly folded monomer and its ability to multimerise.
The results showed all changes resulted in cell lysis as having previously been demonstrated infection of BHK-21 cells with BUNV and in either reduced or ablated viral viability. Interestingly, one of the mutants, E3A, exhibited high levels of gene expression but was unable to induce cell lysis.
This work deepens our understanding of the BUNV multiplication cycle as well as provides a valuable tool for further research towards vaccines and targets for potential anti-viral therapies against pathogens of this order.