Approaches to pseudotype DNA and RNA viruses of veterinary interest for use as tools in cell tropism and immune response studies

Pseudotype viruses (PVs) provide an alternative platform to wild type viruses since they mimic their receptor binding and entry processes and able to interact with the host immune response. For this thesis, a PV system was developed to conduct experimental research on a

DNA and RNA viruses of veterinary interest. Equid herpesvirus 1 (EHV-1) is a DNA virus of the Herpesviridae family representing a significant welfare issue in horses and a great impact on the equine industry. EHV-1 entry mechanism is complicated by the presence of twelve glycoproteins on the viral envelope. To

investigate EHV-1 entry, a simplified lentiviral PV system was generated including gB, gD, gH and gL able to transduce target cell lines demonstrating that the four essential entry glycoproteins of EHV-1 are not only required but also sufficient for cell entry. Successful generation of an EHV-1 PV permitted the development of a PV neutralisation assay (PVNA).

Feasibility of the PVNA was first tested by measuring the level of neutralising antibodies in EHV-1 experimentally infected horses in a longitudinal manner and secondly by detecting an immune response in vaccinated horses. The performance of the assay was compared with a conventional EHV-1 virus neutralisation (VN) assay, showing a good correlation. Furthermore, PVs routinely require -80°C for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, freeze-drying (lyophilisation) of EHV-1 PVs was investigated to address this

issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Influenza viruses are RNA viruses of the Orthomyxoviridae family and some novel Influenza. A viruses that circulate among avian species pose a concern to public health. Partial genomic RNA sequences from an apparently novel strain (denoted Kz52) close to the H9 subtype were isolated from a dead Aythya farina near the Caspian Sea in Kazakhstan. Different approaches were attempted to generate Kz52 PV for the future perspective to develop a PVNA system for screening and sero-surveillance studies in wild birds. Despite the challenges in generating the PV system, here we show the ability of Kz52 HA to cross-react to antibodies suggesting the potential of Kz52 HA to trigger an immune response.