Conferencia online "Innovación y Propiedad Intelectual: retos en el momento actual". Con la participación del expositor Mario Matus, Director Adjunto de la Organización Mundial de la Propiedad Intelectual (OMPI) con sede en Ginebra.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) was declared to be a pandemic and a public health emergency by the World Health Organization in March of 2020. Researchers and medical professionals worldwide have been working nonstop to better understand the disease process of COVID-19 in order to refine treatment protocols and create effective immunizations. Within the pandemic, children make up a unique patient population as they have shown to have similar but less severe clinical features when compared with infected adults. Most pediatric cases of COVID-19 have been reported as asymptomatic or mild with only 8 per 100,000 in the US requiring hospitalization between March 1-July 25, 2020, approximately 576 patients. The case presented is of a 4-year-old Caucasian female with an atypical presentation of a COVID-19-associated pneumonia, with a review of her presentation to the hospital, treatment plan, and discharge. The current frequency of pediatric cases of COVID-19 with severe disease is low, and thus, not fully understood. This case provides an example of successful diagnosis and in-patient treatment, and broadens the scope of severe disease potential from COVID-19 in the pediatric population.

Coronaviruses (CoVs) comprise a family of enveloped viruses that cause respiratory disease in humans, including CoV disease 2019 (COVID-19), caused by severe-acute respiratory syndrome CoV-2 (SARS-CoV-2). For CoV infection to occur, the CoV spike (S) protein must mediate fusion between the viral and host membranes. This entry process can also be repurposed during infection to promote cell-to-cell fusion, further contributing to viral spread. To trigger fusion, S must bind its cognate receptor and be cleaved by host proteases. Identifying cellular proteases capable of triggering CoV fusion is critical to understand CoV entry, tropism, and cell-cell spread, however the range of proteases capable of promoting CoV fusion has not been fully explored. Here, using fusion and entry assays, I provide evidence implicating matrix metalloproteinase-9 (MMP-9) as a fusion trigger for SARS-CoV-2 and HCoV-229E. Additionally, I show MMP-9 expression is upregulated during CoV infection, highlighting its potential relevance as a CoV triggering factor.

Au cours des deux dernières décennies, il a été démontré que les maladies émergentes et réémergentes sont liées à la santé humaine, animale et environnementale. Les infections zoonotiques sont reconnues comme étant responsables d'au moins 75 % des éclosions d'agents pathogènes dans le monde. Certains virus peuvent muter et infecter un large éventail d'hôtes, qui se propagent parmi les humains et entraînent des épidémies ou des pandémies. Les chauves-souris sont connues pour être les mammifères les plus diversifiés géographiquement et le plus répandu sur Terre et peuvent être trouvées à l'intérieur structures abandonnées, ainsi que de grottes. Au Canada, il existe dix-huit espèces de chauves-souris insectivores et huit d'entre elles se perchent au Québec. Elles peuvent loger beaucoup de pathogènes pendant l'hibernation en raison de leur métabolisme distinct. Elles peuvent aussi excréter beaucoup de particules virales par différentes voies telles que la salive, les excréments et l'écholocation. Le but de ce projet de maîtrise est de caractériser les virus que les chauves-souris pourraient potentiellement transporter au Canada, en mettant l'accent sur la détection des Coronavirus. Le premier objectif est de traiter 250 échantillons (matières fécales et organes) prélevés sur des chauves-souris sauvages, puis d'utiliser d'autres techniques biomoléculaires comme le NGS pour détecter un large spectre de virus chez ces chauves-souris. Les échantillons ont été dépistés pour les Coronavirus et les Rhabdovirus à l'aide de la PCR conventionnelle. La prévalence des Coronavirus chez les chauves-souris semble actuellement être relativement faible au Canada et de nombreux facteurs, y compris le petit nombre d'échantillons prélevés jusqu'à présent, pourraient y avoir joué un rôle. Il serait important de continuer à examiner les échantillons de chauves-souris à plus grande échelle afin de caractériser pleinement les viromes que ces animaux hébergent, pour fournir un avertissement des menaces d'épidémie ou de pandémie à la première occasion. / Over the past two decades, emerging and re-emerging diseases have been shown to be interlinked between human, animal, and environmental health. Zoonotic infections are recognized to be responsible for at least 75% of pathogen outbreaks in the world. Certain viruses can mutate and infect a wide range of hosts, which spread amongst humans and lead to epidemics/pandemics. Bats are known to be the most geographically diverse and widespread mammal on Earth and can be found inside of buildings and houses or abandoned structures, as well as caves. In Canada, there are eighteen species of insectivorous bats and eight of them roost in Québec. They can also harbor plenty of pathogens during hibernation due to their distinct metabolism. They can shed plenty of pathogens through different pathways such as saliva, excreta, and echolocation. The overall goal of this master's project is to characterize the pathogens that bats could be potentially carrying in Canada, with a focus on Coronavirus detection. The first objectiveis to process 250 samples (feces and organs) collected from wild bats in the field in Canada and then using other biomolecular techniques such as NGS to detect for the presence of other bat pathogens. The samples were screened for Coronaviruses and Rhabdoviruses using conventional PCR with no positive results. The prevalence of Coronaviruses in bats currently appear to be relatively low in Canada and, many factors, including the moderately low numbers of samples collected in Canada so far, may have played a role. It will be important to continue collecting and screening bat samples on a larger scale to fully characterize the viromes that these animals harbour, to provide warning of epidemic/pandemic threats at the earliest opportunity

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 168-210). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Yunjeong Kim / Coronaviruses are pathogens that cause diverse diseases in humans and animals. The studies in this dissertation are focused on feline coronavirus (FCoV), ferret coronavirus (FRCoV) and mink coronavirus (MCoV). FCoV and FRCoV infections typically cause enteritis in cats and ferrets, respectively. However, a 100% fatal systemic disease called feline infectious peritonitis (FIP) can develop in some FCoV infected cats and a fatal systemic disease resembling FIP can develop in some FRCoV infected ferrets. MCoV causes enteritis which results in significant economic loss to mink farmers. No effective vaccine or treatment is available despite the increasing importance of these viral diseases. We have previously reported the synthesis of inhibitors against 3C-like protease (3CLpro) of FCoV and demonstrated the antiviral efficacy of a 3CLpro inhibitor for treating FIP. FRCoV and MCoV 3CLpro are closely related to FCoV 3CLpro. Therefore, we investigated the structure-function relationships of our 3CLpro inhibitors to identify the struc-tural requirements of inhibitors for FRCoV and MCoV. This is the first report of antiviral com-pounds against FRCoV and MCoV. We have previously conducted a field trial with a potent 3CLpro inhibitor, GC376, in cats with naturally occurring FIP. Comparison of the FCoV 3CLpro amino acid sequences from the pre- and post-treatment samples in one cat showed amino acid changes in 3CLpro. Hence, we generated recombinant 3CLpros carrying the amino acid changes and characterized the effects of these amino acid changes in FCoV 3CLpro on its susceptibility to GC376. We observed that these amino acid changes did not markedly affect the activity of GC376 in fluorescence resonance energy transfer (FRET) assay, explaining the absence of clinical drug resistance in this cat during the field trial.

Les différents génotypes de Coronavirus canins (CCoV-I/II) et félins (FCoV-I/II) sont phylogénétiquement proches, suggérant des transmissions inter-espèces entre chiens et chats. Lors d’analyses de séquences menées sur des chats infectés, des souches félines atypiques ont pu être mises en évidence, contenant un gène S de type FCoV-I, un gène N de type CCoV-I, ainsi que la présence du gène ORF3, spécifique à CCoV-I. Dans ces souches, le gène ORF3 est présent avec une ou deux délétions toujours identiques, conduisant à la synthèse de protéines tronquées gp3-Δ1 et gp3-Δ2. Les délétions de protéines accessoires étant déjà impliquées dans les transmissions inter-espèces, une étude de caractérisation de la protéine gp3 et de ses différentes formes a été menée. Les trois protéines s’oligomérisent de manière covalente et sont retenues dans le réticulum endoplasmique, en absence de signal spécifique de rétention. Les délétions influencent le niveau d’expression des protéines en cellules félines, où seule l’expression de gp3-Δ1 est visible, alors qu’elles conservent toutes une expression optimale en cellules canines. En l’absence de souches de Coronavirus cultivables en laboratoire contenant le gène ORF3, des cellules canines exprimant l’une des protéines gp3 ont été infectées par une souche CCoV-II. Dans ce modèle, les protéines gp3 ne modifient pas le cycle viral. Dans un contexte d’émergence de nouveaux Coronavirus, la compréhension des mécanismes moléculaires de changement d’hôte est cruciale et les Coronavirus félins et canins peuvent représenter un modèle d’étude utile. / The different genotypes of canine (CCoV-I/II) and feline (FCoV-I/II) Coronaviruses share a close phylogenetic relationship, suggesting inter-species transmissions between cats and dogs. Through sequence analyses of cat samples, atypical FCoV strains, harbouring an S gene related to FCoV-I, an N gene close to the CCoV-I cluster and the ORF3 gene, peculiar to CCoV-I, were discovered. This ORF3 gene was systematically truncated in feline samples, displaying either one or two identical deletions, leading to the translation of gp3-Δ1 and gp3-Δ2. As deletions in accessory proteins have already been involved in host-switch, studies of the different variants of gp3 were conducted. Results demonstrate that all proteins oligomerize through covalent bonds and are retained in the ER, without any specific retention signal. Deletions influence the expression level with a proper expression of the three proteins in canine cells, whereas only gp3-Δ1 expression is sustained in feline cells. As no isolates of Coronavirus harbouring the ORF3 gene exists, cells expressing the different gp3 proteins have been infected with a CCoV-II strain. In this model, the gp3 proteins do not influence the viral life cycle. In the light of emergence of new Coronaviruses, investigations on their molecular mechanisms during the host-switch are crucial and canine and feline Coronaviruses could represent a useful model.

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.