Alphaviruses of the family, such as chikungunya virus (CHKV), Semliki Forest virus, Ross River virus and Mayaro virus, cause similar clinical symptoms, usually fever and long-lasting arthralgia. their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives. family, genus and [104,105]. Alphaviruses of the family, such as KMT3A chikungunya virus (CHKV), Semliki Forest virus, Ross River virus and Mayaro virus, cause similar clinical symptoms, usually fever and long-lasting arthralgia. The pseudotyped LV platform not only offers the advantage of a lower containment level (e.g., CHKV is classified as hazard group 3) [88,106,107], but has also been expanded to include multiple alphavirus PVs to distinguish between alphaviruses reactivities . This type of multiplex assay can also be used to isolate cross-reactive neutralising antibodies and to produce broad-spectrum monoclonal antibodies as therapeutics, or to identify critical conserved epitopes to guide vaccine design. A similar approach was proposed by Luczkowiak and colleagues, by using a filovirus pseudotyped LV to investigate the cross-neutralising activity of convalescent sera from Ebola virus disease recovered patients against other members of the genus genus such as West Nile virus (WNV) and Japanese encephalitis virus . The issue lies in the virus life cycle; these are internally budding viruses that acquire their envelope lipidic membrane from the cytoplasmic membrane system (endoplasmic reticulum, Golgi apparatus), whilst the main site of budding for retroviruses and VSV is the plasma membrane. As such, the Envs of internally budding viruses are not in the required location (cell membrane), where the retroviral or VSV particles bud. Research into other platforms has been undertaken to establish alternative backbones for internally budding viruses. Pierson et Bifenazate al. produced a sub-genomic replicon with the non-structural proteins of WNV incorporating a GFP or luciferase reporter gene. When complemented in trans with plasmids expressing WNV structural proteins, single-round replication reporter virus particles (RVP) were generated. These RVP were able to incorporate multiple strains of WNV . Similar approaches have been developed using dengue virus reporter replicons to produce particles with multiple heterologous flavivirus Envs on their surface [171,172]. The uptake of these new systems gives way to a whole new era of pseudotyping. 6. Conclusions Over the course of Bifenazate the last two decades, pseudotyped LVs have cemented themselves as a robust alternative to handling live virus isolates for serological investigations. Studies have exploited unique flexibilities in their design and construction to advance knowledge in challenging areas, overcoming constraints on resources, the need for high-containment facilities or the lack of cell culture systems. While taking note of where we stand today, it is evident that there is an increasing uptake of pseudotyped LVs; as exemplified by the record number of studies throughout 2020 which have widely reported their use in response to the emergence of SARS-CoV-2. We foresee there will be a benefit and need for enhanced characterisation of pseudotyped LV stocks in support of serological investigations supporting medicinal product licensure. Further, enhanced methods of quantification will play a role in comparisons against other novel vector systems, assisting identification of the most appropriate platform for each Env. LVs will act as a prototype in this continued trend towards pseudotype-based serology. Author Contributions Writingoriginal draft preparation (K.T., E.M.B., G.M.); writingreview and editing (K.T., E.M.B., G.M.). All authors have read and agreed to the published version of the manuscript. Funding K.T.s studentship is funded Bifenazate by the National Institute for Biological Standards and Control. E.M.B. is funded by the Coalition for Epidemic Preparedness Innovations. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement No new data were created or analysed in this study. Data sharing Bifenazate is not applicable to this article. Conflicts of Interest The authors declare no conflict of interest. Footnotes Publishers Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations..