Genomic characterization and epidemiological Profile of COVID19 in Rwanda

A: Background Evidence reveals that COVID-19 outbreak is of zoonotic origin, specifically bats seem to be the reservoir of COVID-19 virus, although the intermediate host(s) has not yet been identified. Corona Viruses (COVs) are enveloped positive-stranded RNA viruses with nucleocapsid. For addressing pathogenetic mechanisms of SARS-CoV-2, its viral structure, and genome must be of consideration. The transmission in Rwanda has been increasing overtime although COVID19 prevention measures and policies are being implemented. Although the WHO guided on diagnostic point of view and key biomarkers have been proposed, there remain gaps in understanding molecular patterns and genetic diversity in COVID 19 patients located in the African region. Besides, major gaps still exist in determining molecular factors influencing the susceptibility and vulnerability to severe coronavirus disease within a specific group of population. B: Goals and Objectives The project aims to determine the genetic pattern and diversity of severe coronavirus infection in infected patients and hence identify the outbreak transmission chain in Rwanda. More significantly, this study will provide understanding of the outbreak evolution in Rwanda and observing transmission dynamics through building phylogenetic trees. The project has the following specific objective: 1. To compare the diagnosis of Covid-19 from positive and control cases in Rwanda using both serological and molecular techniques and establish the relationship between the SARs-Cov-2 viral load and disease outcome 2. To determine the sequences of the Covid-19 positive cases reported in Rwanda and corelate with disease outcome 3. To determine the expression of the ACE-2 gene and protein receptor in the Covid-19 positive and control cases and corelate to clinical data 4. To perform protein-protein interaction analysis of the SARs-Cov-2 and ACE-2 receptor and truncated domains and establish its value for use as a diagnostic biomarker C: Methods Research methods: The study will employ a cross-sectional survey design that will target all Covid-19 confirmed or suspected cases treated in Rwanda at the sampling time. • Clinical Specimen and RNA extraction: Nasopharyngeal or oropharyngeal swab which were collected from symptomatic and symptomatic patients to detect SARS-CoV-2 by real-time reverse transcriptase (RT)-PCR will be used for RNA extraction with Biamp viral RNA mini kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. All specimens will be handled under a biosafety cabinet according to laboratory biosafety guidelines of Rwanda National Reference Lab. • Sequencing analysis: Using reverse transcriptase, cDNA will be synthesized from RNA extracted from the cultured cell medium in which the virus will be replicated. For this project the Whole Genome Sequencing will be performed using Illumina Mini-Seq machine 18 • Data Quality Assessment: Investigating quality control of sequence reads using FastQC. Fast QC is a Java-application that provides quality checks on raw sequence data coming from highthroughput sequencing pipelines. To improve the quality of data reads below an average score of 20 bp will be discarded for contamination. The reads will be trimmed using Trimmomatic. Trimmomatic is a flexible pipeline tool used for quality filtering. • Alignment and Assembling: Aligning the reads to a reference genome using a three-step process. Assembling and merging the read transcripts with an annotated file of the reference genome to produce a single annotation file using Cuffmerge. D: Expected outcomes The findings of the project will have the following clinical implications: • Screening the Covid-19 positive and suspected cases by both molecular and serological techniques would inform of the diagnostic ability of each of the tests and provide insight into the ability of each test to predict severe disease and guide into the triaging of patient • Determining the association between the viral load and disease severity would guide towards the evaluation of the management approaches and ensure that only very severe cases are managed by hospitalization while mild cases can be monitored at home or at outpatient facilities • Determining the SARs-Cov-2 recombination/mutation events by sequence analysis would inform of the disease transmission patterns and guide towards the adoption of management practices that are tailored as per the observed transmission patterns. • Assessing the ACE-2 levels in the Covid-19 samples in Kenya would inform of the role of this receptor in modulating viral infectivity and subsequently establish the susceptibility of the Kenyan population to Covid-19. Corelating the expression of this receptor with the patient clinical data would inform of the therapies to foster especially in patients with preexisting conditions. In addition, evaluating the role of this protein as a prognostic indicator would provide insight into the development of ideal point of care diagnostics to fast track disease detection. • Research outputs shall be communicated via scientific conferences and policy reports and subsequently published in open access peer reviewed international journals to inform a wider scientific community The key expected outcomes: • COVID19 transmission in Rwanda identified • New spots for vaccine and therapeutic development and hence contribute to the global efforts • Evidence on genetic diversity for COVID19 in African will be generated