Development and lyophilisation of influenza, rabies and filovirus lentiviral pseudotypes for use in neutralisation assay-based diagnostic kits.

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates ?80?C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5 M sucrose–PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37?C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. Further to this, the ongoing outbreak of Ebola virus in West Africa has exacerbated the need for assays that will permit the virus to be studied more widely in low containment laboratories. To address this we have generated a panel of Ebolavirus PVs and shown them to be sensitive to neutralisation by monoclonal antibodies. These results confirm the viability of a freeze-dried PVNA-based kit and suggest filovirus PV are a suitable source of antigen for neutralisation assays. This could significantly facilitate low-cost, widely applicable serology for a wide portfolio of emerging infectious viruses (www.viralpseudotypeunit.info).