Production of Molecular Based Point-of-care Protocol for SARSCoV-2 Detection among Humans, and Comprehensive Atlas of Possible Coronavirus Immune Response Differences Between Humans, and Non-Human Primates.

A: Background: Covid-19 has spread to almost all countries of the world and caused massive losses both in health and economic viewpoints. Apart from humans, scientific evidence also shows that primates are very sensitive to human diseases. Mountain gorillas for instance are prone to some respiratory illnesses that afflict humans. A common cold can kill a gorilla. In Rwanda, where tourism is an important source of revenue, and the government has prioritized the protection gorillas during COVID-19 with restrictive measures, there is no scientific evidence available about any potential transmission of COVID-19 from humans to gorillas or vice-versa. In addition, the adequacy of immunological defense mechanism in gorillas has not been tested yet. Some scientific experts are indicating that primates, including mountain gorillas, are likely susceptible to complications arising from the COVID-19. Today, more than 600 mountain gorillas live in Virunga massif where Volcanoes National Park is located. Not only many primate populations are already endangered, but also a spread of COVID-19 could put them in even more danger. B. Goal and Objectives The main goal of this study is twofold; first, to produce a Comprehensive Atlas of Possible Coronavirus Immune Response Differences Between Humans, and Non-Human Primates; second, in collaboration with our partners, to produce a molecular-based point-of-care protocol for SARS-CoV-2 detection. Objective 1: The Humans' sample (from recovered, active, and naive), as well as non-human primates samples (from chimpanzees, mountain gorillas, baboons, golden monkey, African green monkeys) testing regime includes: • To assess the production of neutralizing antibodies among different groups of humans and nonhuman primates, • To stimulate the T cells and assess their activities (activation markers expression, production of cytokines); • To document a scientific proof of respiratory infections among the non-human primates' routes of transmission; • A possible functional comparison of SARS-CoV-2 immune responses pathways (susceptibility, protective immunity) between humans, and non-human primates; • Possible epidemiology of SARS-CoV-2 in different selected non-human primates' reservoirs. 9 Objective 2: The detection protocol includes the following: • A point-of-care (hardware) instrument that is fit for purpose. • A biological assay that includes a simple extraction (heat and chill) protocol and fluorescent-dye based detection of Covid-19. • A software capable of depositing the test results into a central database from distant geographies where the assay is performed into a secure central database. i) The first stage involves testing primers/probes against a synthesized and/or cloned virus target into a vector. ii) The second stage will include testing of deactivated viral titres from a Guthrie card. The main reason for this kind of testing stage is to control and optimize the nucleotide extraction method before the isothermal PCR detection protocol. iii) The third and final test run will be testing clinical samples for both the extraction method and isothermal PCR detection to assess the functionality of the assay within various matrices applicable for SARS-CoV-2 testing. D: Expected Outcomes are: • A comprehensive atlas of possible SARS-CoV-2 immune response differences between humans, and non-human primates; • A thorough assessment of mountain gorilla's susceptibility to SARS-CoV-2; • A protocol for recommendation for preventing nonhuman primates given the current covid-19 outbreak; • A T16 LAMP instrument capable of conducting Covid-19 assays at point-of-care; • A LAMP based functional assay that can detect SARS-CoV-2; A B C D A: Mountain gorilla physical Examination; B: Sampling; C-D: RNA extraction process of nonhuman primates' samples at Wildlife Virology laboratory- Rubirizi, Kigali Methods: Isolated strains will be sequenced using the Next Generation Sequencing. Obtained sequences will be analyzed and compared to several other strains identified in other places. Amplicons will be prepared for sequencing. The detection protocol will include the use of a molecular diagnostic approach for COVID-19 diagnosis using realtime loop-mediated isothermal amplification (RT-LAMP). Here there will be three sets of virus and virus "equivalents" testing. 10 • An IT system based on a web application that can transmit test results from the field to a central database.