Rational approaches for attenuation of a segmented genome RNA virus

Vaccines are among the most effective means to preventing diseases. With regard to viral diseases both killed and live vaccines are available to prevent and control a number of human and animal viral infections, though for a significant number of diseases, particularly those caused by emerging viruses, no vaccines have yet been developed. Live vaccines comprise modified viruses that have reduced capacity to cause disease i.e. they are attenuated. An advantage of live-attenuated vaccines is that they induce complex immune responses that mimic those of an authentic infection. Potential disadvantages of live-attenuated viruses include the possibility of disease in some recipients (e.g. the immunocompromised), greater instability compared to killed vaccines, and changes to the vaccine that result in restoration of the disease-causing ability of the virus. In addition, there is the possibility for the attenuated virus to interact with circulating wild type, or closely related, strains, also resulting in restoration of virulence. For viruses which have their genetic information separated on different segments, such as influenza virus, this could occur by mixing (reassortment) of genome segments in dually infected cells, resulting in progeny viruses with segments derived from each the two parent viruses. The aim of this project is to manipulate the genome of a model segmented genome virus, Bunyamwera virus, by recombinant DNA techniques, to produce an attenuated virus that is unable to reassort with another viral strain. Proof-of-principle could lead to the development of safer live attenuated vaccines for segmented genome viruses.

The family Bunyaviridae comprises more than 350 viruses characterised by a tri-partite, negative-sense RNA genome. The family includes a number of significant human and animal pathogens and provides many examples of emerging viruses. By reverse genetics we created a recombinant Bunyamwera virus (BUNV) called BUNdelNSs that is unable to block the host innate immune response and is attenuated in cells with a competent interferon system. Viruses with targeted deletions in their IFN-antagonistic functions are excellent candidates for live virus vaccines. They can be grown to high titres in IFN-deficient cell cultures but are attenuated in vivo since they elicit robust innate and adaptive immune responses. While BUNdelNSs could be a potential live attenuated vaccine, there exists the possibility of genome segment reassortment with wild type virus, resulting in restoration of virulence. The aim of the project is to generate versions of BUNdelNSs virus that are unable to reassort their genome segments with isogenic wild type BUNV (or indeed related orthobunyaviruses). Gross sequence changes will be incorporated into the non-coding sequences (which contain cis acting sequences for replication) of each segment to produce debilitated viruses. By serial passage of such viruses in tissue culture we will test the hypothesis that forced evolution will improve the fitness (replication efficiency) of the viruses, and in turn that these evolved viruses will no longer be able to reassort with wild type viruses. We will also attempt to create recombinant viruses containing bipartite or monopartite genomes, again predicting that such viruses would be genetically inert to reassortment with wild type BUNV. If proof-of-principle can be obtained, similar approaches could be used to develop live-attenuated vaccines against the serious pathogens in the family.