Coronaviruses have caused high pathogenic epidemics within the human population on two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-MERS). Four other species of coronavirus circulate endemically in the human population (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics can be used to study an organism’s phenotype by logical mutation of its genotype. Construction of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This system should aid in answering remaining questions of coronavirus genetics and evolution as well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS. / October 2015

Atrial fibrillation, long QT syndrome, and Middle East Respiratory Syndrome are three deadly human diseases for which genetic animal models are needed. From elucidating disease pathogenesis to facilitating the development of treatments, animal models are crucial for studying human disease. One of the most effective ways to generate specific animal models is through genetic modification. Historically, mice have been most widely used as genetically modified models, despite a number of limitations. New gene editing technologies such as CRISPR/Cas9 have made developing alternative genetic models that better recapitulate some human diseases better and more feasible. In this thesis, I describe my efforts to develop genetically modified goat and hamster models for atrial fibrillation and long QT syndrome, and genetically modified hamster models for Middle East Respiratory Syndrome. For long QT syndrome model development, I knocked out the KCNQ1 gene in goat fetal fibroblast cells and baby hamster kidney cells using the CRIPSR/Cas9 system. The knockout results in loss-of-function mutations, a known cause of human long QT syndrome. The edited goat fibroblast cells will be nuclear donors for future cloning experiments to produce live goats possessing the KCNQ1 knockout. The CRISPR gene targeting sgRNA/Cas9 vector, specific for the hamster KCNQ1, has been used for pronuclear injections to produce KCNQ1 knockout hamsters. For atrial fibrillation model development, I designed a single-stranded donor oligonucleotide that generates a KCNQ1 gainof-function mutation resulting in the disease. This oligonucleotide was injected into hamster embryos along with the KCNQ1 sgRNA/Cas9-expressing vector to generate hamsters containing the gain-of-function mutation. Finally, for Middle East Respiratory Syndrome model development, I established a breeding colony of human DPP4 transgenic hamsters in the STAT2 knockout background. Human DPP4 transgenic hamsters are susceptible to MERS-CoV infection, showing mild clinical signs and allowing viral replication in lung tissue. Giving these hamsters a STAT2 knockout background should promote a more severe disease progression. For all three diseases, the foundations for the development of genetic animal models have been laid.

Introduction: Only 3 types of coronavirus cause aggressive respiratory disease in humans (MERS-Cov, SARS-Cov-1, and SARS-Cov-2). It has been reported higher infection rates and severe manifestations (ICU admission, need for mechanical ventilation, and death) in patients with comorbidities such as diabetes mellitus (DM). For this reason, this study aimed to determine the prevalence of diabetes comorbidity and its associated unfavorable health outcomes in patients with acute respiratory syndromes for coronavirus disease according to virus types. Methods: Systematic review of literature in Pubmed/Medline, Scopus, Web of Science, Cochrane, and Scielo until April of 2020. We included cohort and cross-sectional studies with no restriction by language or geographical zone. The selection and extraction were undertaken by 2 reviewers, independently. The study quality was evaluated with Loney’s instrument and data were synthesized by random effects model meta-analysis. The heterogeneity was quantified using an I2 statistic. Funnel plot, Egger, and Begg tests were used to evaluate publication biases, and subgroups and sensitivity analyses were performed. Finally, we used the GRADE approach to assess the evidence certainty (PROSPERO: CRD42020178049). Results: We conducted the pooled analysis of 28 studies (n = 5960). The prevalence analysis according to virus type were 451.9 diabetes cases per 1000 infected patients (95% CI: 356.74-548.78; I2 = 89.71%) in MERS-Cov; 90.38 per 1000 (95% CI: 67.17-118.38) in SARS-Cov-1; and 100.42 per 1000 (95% CI: 77.85, 125.26 I2 = 67.94%) in SARS-Cov-2. The mortality rate were 36%, 6%, 10% and for MERS-Cov, SARS-Cov-1, and SARS-Cov-2, respectively. Due to the high risk of bias (75% of studies had very low quality), high heterogeneity (I2 higher than 60%), and publication bias (for MERS-Cov studies), we down rate the certainty to very low. Conclusion: The prevalence of DM in patients with acute respiratory syndrome due to coronaviruses is high, predominantly with MERS-Cov infection. The unfavorable health outcomes are frequent in this subset of patients. Well-powered and population-based studies are needed, including detailed DM clinical profile (such as glycemic control, DM complications, and treatment regimens), comorbidities, and SARS-Cov-2 evolution to reevaluate the worldwide prevalence of this comorbidity and to typify clinical phenotypes with differential risk within the subpopulation of DM patients. / Revisión por pares