Recovery of an African horsesickness virus VP2 chimera using reverse genetics

Abstract

African horsesickness (AHS) is a devastating disease which causes mortality in more than 95% of fully susceptible horses. There are nine serotypes of the etiological agent, African horsesickness virus (AHSV) exhibiting different levels of cross protection. For an animal to be fully protected against disease, it must be immune against all serotypes. The current live-attenuated polyvalent vaccine produced in South Africa is effective, but a gap exists in the immunological protection of the horse population due to the absence of serotypes five and nine from the vaccine. Reverse genetics systems for AHSV and BTV have led to the development of next generation candidate Disabled Infectious Single Animal (DISA) vaccine platforms. The rescue of DISA vaccines for different BTV serotypes using reverse genetics derived BTVs expressing chimeric VP2 that include regions originating from two different serotypes, has shown that these BTV chimeras can be neutralised by antisera from both serotypes. This project explored the possibility of incorporating antigenic regions of more than one serotype into a common attenuated AHSV4 (AHSV4LP) backbone, by exchanging and replacing the central and tip domains of AHSV4LP VP2 with that of either AHSV5 or AHSV6. The protein structure of VP2 of AHSV was used as a basis for the rational structure-based design of the chimeric AHSV VP2 proteins. Chimeric genome segment 2 plasmid constructs were generated to harbour the coding regions of the central and tip domains of either AHSV5 or AHSV6 in the AHSV4LP backbone. The designed chimeric viruses (rAHSV4-5cVP2 and rAHSV4-6cVP2) were shown to be infectious as evidenced by the rescue of infectious virus upon transfection with expression plasmids and (+)ssRNA T7 transcripts into permissive BSR-T7/5 cells. The recovery of virulent rAHSV5 (FR) entirely from plasmids was also demonstrated in this study. This plasmid based RG system proved to be not only less time consuming but considerably less expensive as it was no longer necessary to synthesise capped (+)ssRNA transcripts in vitro. Agarose gel electrophoresis, real-time RT-PCR and MiSeq sequencing was performed on the rescued chimeric viruses to confirm their identity. All the results and data obtained from the rAHSV4-5cVP2 virus confirmed that the CPE observed in cell culture post-transfection was indeed due to infection with the designed rAHSV4-5cVP2 chimeric virus. The results pertaining to the rAHSV4-6cVP2 virus were not as straight forward. It was shown that a 1400bp deletion had taken place in genome segment 2, but a chimeric genome segment 2 containing regions originating from serotype 4 and 6 was still present at least in a small part of the virus population. Furthermore it was determined that a mutation in the form of a T409bp insertion (in genome segment 2) had taken place in majority of the rAHSV4-6cVP2 virus population which in all likelihood caused the early termination of the open reading frame of VP2. Virus neutralisation assays on the three parental serotypes rAHSV4LP, rAHSV5 (FR) and AHSV6 and the chimeric viruses, rAHSV4-5cVP2 and rAHSV4-6cVP2, demonstrated that rAHSV4-5cVP2 was neutralised by antisera raised against the AHSV4LP backbone, as well as AHSV5. The neutralisation tests of rAHSV4-6cVP2 could not with confidence demonstrate that the exchange of the central and tip domains with serotype 6 changed the antigenicity of the virus, even though neutralisation was observed. In summary, this study demonstrated for the first time that it is possible to generate infectious AHSV with chimeric VP2 using the established reverse genetics system for AHSV4LP. The study also demonstrated that virulent rAHSV5 (FR) could be rescued using an entirely plasmid based reverse genetics system. Furthermore the study demonstrated that the exchange of the coding regions of the central and tip domains of VP2 of AHSV4LP with another serotype (AHSV5) changed the antigenicity of the resulting chimeric virus. Furthermore this study also provided the first experimental data that the exposed central and tip domains of AHSV VP2 are partially responsible for the determination of serotype-specificity but that other regions of VP2 are also involved. The ability to now recover AHSV VP2 chimeras has established a platform to not only develop vaccine candidates against multiple serotypes with single viruses but also provides a means by which to investigate how changes in genome segment 2 sequences affect the resulting VP2 structure and subsequent viral properties