Environmental science

Since the creation of the Global Polio Eradication Initiative (GPEI) in 1988, significant progress has been made toward attaining a poliovirus-free world. This has resulted in the eradication of wild poliovirus (WPV) serotypes two (WPV2) and three (WPV3) and limited transmission of serotype one (WPV1) in Pakistan and Afghanistan. However, the increased emergence of circulating vaccine-derived poliovirus (cVDPV) and the continued circulation of WPV1, although limited to two countries, pose a continuous threat of international spread of poliovirus. These challenges highlight the need to further strengthen surveillance and outbreak responses, particularly in the African Region (AFRO). Phylogeographic visualization tools may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance. We created a comprehensive protocol for the phylogeographic analysis of polioviruses using Nextstrain, a powerful open-source tool for real-time interactive visualization of virus sequencing data. It is expected that this protocol will support poliovirus elimination strategies in AFRO and contribute significantly to global eradication strategies. These tools have been utilized for other pathogens of public health importance, for example, SARS-CoV-2, human influenza, Ebola, and Mpox, among others, through real-time tracking of pathogen evolution (https://nextstrain.org), harnessing the scientific and public health potential of pathogen genome data.

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production of transcription- and replication-component virus-like-particles (trVLPs) by genetic transcomplementation. The system consists of two parts: SARS-CoV-2 GFP/ΔN genomic RNA, in which the nucleocapsid (N) gene, a critical gene for virion packaging, is replaced by a GFP reporter gene; and a packaging cell line for ectopic expression of N (Caco-2-N). The complete viral life cycle can be recapitulated and confined to Caco-2-N cells, with GFP positivity serving as a surrogate readout for viral infection. In addition, we utilized an intein-mediated protein splicing technique to split the N gene into two independent vectors and generated the Caco-2-N^intein cells as a packaging cell line to further enhance the security of this cell culture model. Altogether, this system provides for a safe and convenient method to produce trVLPs in BSL-2 laboratories. These trVLPs can be modified to incorporate desired mutations, permitting high-throughput screening of antiviral compounds and evaluation of neutralizing antibodies. This protocol describes the details of the trVLP cell culture model to make SARS-CoV-2 research more readily accessible.