Since its emergence in December 2019, SARS-CoV-2 has infected over 200 million people globally. SARS-CoV-2 spike (S) decorates the viral envelope and is responsible for facilitating viral entry into the host cell. To mediate membrane fusion, S must be proteolytically cleaved. For the closely related SARS-CoV S, cleavage at the host cell surface must be facilitated by the serine protease TMPRSS2. We demonstrated that SARS-CoV-2 S can facilitate fusion independent of TMPRSS2 and sought to identify other proteases capable of driving SARS-CoV-2 S-mediated fusion. We show that the ADAMs and MMP inhibitor GI 254023X is capable of substantially reducing SARS-CoV-2 S-mediated syncytium formation. Additionally, we identified MMP-9, a protein target of GI 254023X, as a host protease capable of enhancing SARS-CoV-2 lentivirus entry in HEK293T-ACE2 cells. These results implicate ADAM and MMP proteases, in particular MMP-9, as potential antiviral drug targets against COVID-19 pathogenesis.

Objetivos: Determinar hallazgos estructurales o funcionales ecocardiográficos en pacientes recuperados de infección SARS-CoV-2. Materiales y métodos: Estudio observacional transversal, que incluyó pacientes trabajadores de un hospital nacional COVID, estudiados entre 3 a 6 meses luego del diagnostico de infección SARS-CoV-2. La exploración ecocardiográfica se desarrolló de forma sistemática e incluyó las modalidades convencionales. Resultados: Se incluyeron 65 casos con infección-CoV-2, la edad promedio fue 37.7 años, la obesidad resultó la comorbilidad mas frecuente (13.8%) y la presentación clínica leve fue la de mayor prevalencia (84.6%). Las medias del diámetro diastólico y la fracción de eyección ventrículo izquierdo fueron 42mm y 57% respectivamente. Así mismo la media del diámetro basal del ventrículo derecho fue de 31mm, de la fracción de acortamiento 44% y en todos los casos se reportó probabilidad de hipertensión pulmonar como baja. No se encontró efusión pericárdica en ninguno de los casos. Conclusiones Los pacientes recuperados de infección SARS-CoV-2, no presentan alteraciones estructurales ni funcionales en la exploración ecocardiográfica convencional.

SARS-CoV-2 wastewater surveillance is a promising tool for monitoring the spread of infection during pandemic outbreaks. 24-hour composite sampling of wastewater using autosamplers is the preferred means for wastewater surveillance sample collection. Autosamplers however require a significant capital cost and furthermore some sampling locations are not amenable to autosampler deployment because of a lack of space and lack of access to electricity. Grab sampling is an alternative to auto sampling for wastewater surveillance, however it may be less effective compared to 24-hour composite sampling due to the possibility to miss the collection of shed disease targets during critical shedding events. Torpedo-style passive samplers packed with medical gauze and tampon-style passive samplers are alternatives to grab sampling when deployment of autosamplers is not possible. Torpedo-style and tampon-style passive samplers are characterized as being easy to deploy and collect and have shown promise for disease surveillance using wastewater. Although passive samplers have shown the ability to detect SARS-CoV-2, they have not demonstrated the ability to quantify the viral load in the wastewater due to the fact that the collection of the liquid phase of the sampler is not consistent across the deployment period of a passive sampler. As SARS-CoV-2 disease targets have been shown to largely partition to the solids phase of wastewaters, it is hypothesized that mass fraction quantitation may enable passive samplers to quantify wastewater signals comparably to autosamplers. In this study, wastewater samples were collected from the same location over a period of three months from a sewer access point at the University of Ottawa using conventional 24-hour auto sampling. Two types of torpedostyle passive samplers and a tampon-style passive sampler were tested to assess whether passive sampler measurements of SARS-CoV-2 N1 and N2 gene targets can be used in the place of autosampler quantitated values. When comparing the wastewater characteristics of centrifuged pellets collected by various passive samplers and a conventional autosampler, the results of this study showed that the torpedo-style passive sampler packed with two pieces of gauze (P2) collected significantly lower water content compared to the autosampler, and P2 collected significantly greater total solids and volatile solids compared to the autosampler. When measuring SARS-CoV-2 N1 and N2 signals, the results indicate that N1 and N2 gene region copy numbers from all of the samplers were not significantly distinct. However, the P2 sampler, a torpedo-style passive sampler packed with four pieces of gauze (P4), and the tampon-style passive sampler (T) captured a greater quantity of pepper mild iii mottle virus (PMMoV) gene targets compared to the autosampler; where PMMoV is the most commonly measured fecal biomarker for wastewater surveillance of SARS-CoV-2. The greater quantity of PMMoV gene targets compared to the autosampler was likely due to proportionally higher total solids and volatile solids in the centrifuged pellet material captured. When N1 and N2 measurements were normalized against sample volume, pellet mass or PMMoV gene copy numbers, P2, P4, and T showed no significant differences compared to the autosampler. In contrast, differences were observed between passive samplers and the autosampler when PMMoV measurements were normalized against the matrix volumes or pellet mass. High statistical percentage differences were observed between all passive samplers and the autosampler. Overall, passive samplers are reliable, cost-effective devices for sampling disease targets in wastewater if results are expressed as copies/g or copies/copies PMMoV. These devices are feasible substitutes for autosamplers when detection and quantification of SARS-CoV-2 in wastewater are required. P2 passive samplers using units of measurement of copies/g are recommended for SARS-CoV-2 surveillance in the wastewater.

SARS-CoV-2 has plagued the world since 2019 with continuously emergence of new variants, resulting in repeated waves of outbreak. Although the countermeasures like vaccination campaign has taken worldwide, the sophisticated virus mutated to escape immune system, threatening the public health. To win the race with the virus and ultimately end the pandemic, we have to take one step ahead to predict how the SARSCoV-2 might evolve and defeat it at the beginning of a new wave. Hence, we proposed a deep learning based framework to first build a deep learning model to shape the fitness landscape of the virus and then use genetic algorithm to predict the high-risk variants that might appear in the future. By combining pre-trained protein language model and structure modeling, the model is trained in a supervised way, predicting the viral transmissibility and antibodies escape ability to eight antibodies simultaneously. The prevenient virus evolution trajectory can be largely recovered by our model with high correlation to their sampling time. Novel mutations predicted by our model show high antibody escape through in silico simulation and overlapped with the mutations developed in prevenient infected patients. Overall, our scheme can provide insights into the evolution of SARS-CoV-2 and hopefully guide the development of vaccination and increase the preparedness.

Background: Respiratory tract infections are responsible for millions of deaths annually. Interferon-stimulated genes (ISGs) play a significant role in fighting off viral respiratory tract infections in the antiviral defence system. Measuring extracellular protein products of ISGs could be potential biomarkers of viral infection. Although, the feasibility and performance of ISGs as functional and robust clinical biomarkers from a non-invasive sample format remains unknown. Methods: Three ISGs, CXCL10, CXCL11, and TNFSF10, were examined in in-vivo and in-vitro gene expression datasets (RNA-sequencing and microarray) infected with common respiratory tract infections (Rhinovirus, Respiratory syncytial virus, influenza A and SARS-CoV-2) samples and compared to negative controls. Using qualitative selection criteria of 1) elevated presence in at least one dataset with viral infection, 2) secreted protein product, and 3) commercially available antibodies for detection, CXCL10, CXCL11 and TNFSF10 gene expression levels were assessed. A correlation analysis was performed with SARS-CoV-2 infection severity and gene expression kinetics. CXCL10 was subsequently validated at the protein level in saliva as a prerequisite for developing a host-response LFA. Results: CXCL10 and CXCL11 upregulation were positively correlated with RSV compared to control (p < 0.05). CXCL10/CXCL11/TNFSF10 were not different between samples collected from RV infected subjects relative to controls (p > 0.05). No significant association was found with influenza A for all three genes. CXCL10/CXCL11/TNFSF10 upregulation was positively correlated with SARS-CoV-2 infection compared to control (p < 0.001). CXCL10 expression correlated with COVID-19 viral load. CXCL10 was chosen as a lead biomarker candidate based on these analyses that included different virus infections, time-courses, and measures of severity. CXCL10 was not detected at the protein level in healthy saliva but was elevated in saliva from COVID-19 patients. A CXCL10 LFA was developed with a sensitivity of 2 ng/ml in a buffer and artificial saliva. Conclusion: We establish and validate the potential of developing rapid test techniques to examine host immune response from a bioinformatic approach to developing a prototype rapid test with capabilities to be used in point-of-care settings. / Thesis / Master of Science (MSc) / Respiratory tract infections are a leading cause of death and one of the main reasons to seek primary care. Both historically and in the present day, respiratory tract infections remain a massive socioeconomic burden. Current diagnostics fail to quickly identify a respiratory tract infection's etiology, and prognosis, leading to suboptimal patient care and the over prescription of antibiotics. Advanced tools used in academia and research, including next-generation -omics sequencing and metagenomics, have capabilities to identify all nucleic acid material in a sample - including host RNA- which offers potential to improve the diagnosing of respiratory tract infections. However, these technologies have not been integrated into routine care due to economic, technical, and logistical barriers. We explored host RNA (transcriptomics), looking at antiviral interferon-stimulated genes for their potential as a biomarker of viral infection amenable to point-of-care testing platforms from non-invasive sample types.

The Coronavirus disease 2019 (COVID-19) pandemic has highlighted the critical need for accurate and sensitive diagnostic tools for detecting the SARS-CoV-2 virus. The nucleocapsid (N) protein is essential for virus replication and plays vital roles in virus assembly, packaging, and RNA transcription. This protein is a crucial component of the viral particle and is less prone to mutations than the other essential proteins in SARS-COV-2. All of these make the N protein a reliable target for virus detection. Aptamers, single-stranded oligonucleotides that can specifically bind to target molecules, have been proposed as a promising alternative to antibodies for detecting and treating viral infections. This study aimed to select DNA aptamers against the N protein of SARS-CoV-2 using capillary electrophoresis (CE) and validate the binding specificity of the aptamers. After selecting seven clones, a preliminary binding validation was performed, and the two best binding clones were identified as ECK4 and ECK6. The structures of the aptamers were then modified by removing the primer regions from the original sequence, and the binding capacity of the truncated aptamers was confirmed. Dissociation constant (KD) values were calculated to provide further supportive information for the quality of the two clones. Additionally, Biolayer interferometry (BLI) was used to calculate Apparent KD as an alternative technique and provided consistent results with CE. Our results demonstrate the successful selection of aptamers for the N protein of SARS-CoV-2 using CE-SELEX. Confirming the aptamers' binding capacity to N protein paves the way for developing aptamer-based diagnostics for COVID-19.

The recent pandemic has reminded the public that basic research in virology is pivotal for human health. Understanding the mechanisms of successful viral replication and the role of host factors can help to combat viral infections and prevent future pandemics. Our lab has published the first SARS-CoV-2 RNA-protein interaction atlas, laying the foundation to investigate the interplay between viral RNA and host RNA binding proteins (RBP). Based on this, my project created the largest collection of binding profiles of host and viral RBPs on SARS-CoV-2 RNA to date. This revealed the host protein SND1 as the first human RBP that specifically binds negative sense viral RNA at the 5´ end, a region associated with viral transcription initiation. The binding profile shares similarities with the viral RBP nsp9, which binds the 5´ ends of positive and negative sense SARS-CoV-2 RNA. Depletion of SND1 shows reduced levels of viral RNA revealing it as a proviral host factor. To decode the underlying molecular mechanism, I characterized the protein-protein interactions of SND1 in SARS-CoV-2 infected and uninfected cells. Infection remodels the protein interactors of SND1 from general RNA biology to membrane association and viral RNA synthesis. Upon infection, SND1 specifically interacts with nsp9, the RBP that shares the same binding region on the negative strand of SARS-CoV-2 RNA. Recent work demonstrates that nsp9 is NMPylated in vitro suggesting a functional role of nsp9 in priming of viral RNA synthesis. I was able to show that nsp9 is covalently linked to the 5´ ends of SARS-CoV-2 RNA during infection of human cells. Analysing the covalent bond of nsp9 with the viral RNA on nucleotide level shows close proximity to the initiation sites of viral RNA synthesis, suggesting that nsp9 acts as a protein-primer of SARS-CoV-2 RNA synthesis. SND1 modulates the distribution of nsp9 on the viral RNA, since depletion of SND1 results in imbalanced occupancy of nsp9 at the 5´ends of viral RNA. This study is the first to provide evidence for the priming mechanism of SARS-CoV-2 in authentic viral replication and further reveals how this mechanism is modulated by the host RBP SND1. Detailed knowledge about priming of viral RNA synthesis can help to find targeted antivirals that could be used to fight coronaviral infections. / Die letzte Pandemie zeigte erneut, das Grundlagenforschung im Bereich der Virologie essentiell für die Gesundheit des Menschen ist. Das Wissen über Schlüsselelemente erfolgreicher viraler Replikation und der Relevanz humaner Proteine darin kann helfen Infektionen zu bekämpfen und künftige Pandemien zu verhindern. Unser Labor publizierte das erste SARS-CoV-2 RNA Protein-Interaktom und legte dabei den Grundstein für die Forschung am Zwischenspiel viraler RNA und humanen RNA Bindeproteinen (RBPs). Basierend darauf, generierte mein Projekt die bislang größte Sammlung an Bindeprofilen humaner sowie viraler RBPs auf der SARS-CoV-2 RNA. Dabei zeigte sich der Wirtsfaktor SND1 als das erste human RBP das in der Lage ist den Negativstrang der viral RNA zu binden, spezifisch an dessen 5´ Ende welches mit der Transkriptionsinitiierung assoziiert ist. Diese Bindestelle ist ähnlich zu dem viralen RBP nsp9, welches die 5´ Enden der positiv und negativ RNA bindet. Das Fehlen von SND1 in der Wirtszelle führt zu reduzierten Mengen viraler RNA und impliziert daher einen proviralen Einfluss von SND1. Um den zugrundeliegenden molekularen Mechanismus zu verstehen, betrachtete ich die Protein-Protein Interaktionen von SND1 in SARS-CoV-2 infizierten und uninfizierten Zellen. Dabei zeigte sich, dass durch die Infektion die Interaktionspartner von SND1 von genereller RNA Biologie zu Membranassoziierung sowie viraler RNA Synthese verschiebt. Mit Infektion der Zelle interagiert SND1 spezifisch mit nsp9, das RBP welches dieselbe Binderegion am Negativstrang mit SND1 auf der SARS-CoV-2 RNA teilt. Neuste in vitro Studien zeigen, dass nsp9 NMPyliert wird und deuten damit eine Relevanz von nsp9 in Priming an. Ich konnte im Kontext authentischer viraler Replikation zeigen, dass nsp9 kovalent an die 5´ Enden der SARS-CoV-2 RNA gebunden ist. Bei genauerer Untersuchung der kovalenten Bindung von nsp9 an der viralen RNA auf Nukleotidebene zeigt, dass diese Nahe der Initiationsstelle der Transkription liegen, was eine Relevanz von nsp9 als Protein-Primer in der SARS-CoV-2 RNA Synthese impliziert. Die Richtige Verteilung von nsp9 auf der viralen RNA wird von SND1 moduliert, da Abwesenheit von SND1 zu einem Ungleichgewicht von nsp9 an den 5´ Enden führt. XII Diese Studie ist die Erste, die Evidenzen für den Primingmechanismus von SARS-CoV-2 in authentischer viraler Replikation zeigt und wie diese durch SND1 moduliert wird. Detailliertes Wissen über das Priming viraler RNA Synthese kann dabei helfen gezielte nach antiviralen Substanzen zu suchen, die dabei helfen könnten Infektionen durch Coronaviren zu bekämpfen.

Editorial

Como parte importante de la sociedad, las universidades forman un grupo activo que trabaja en la investigaciòn y el desarrollo de iniciativas que combaten el COVID-19

Background: Cancer patients are at higher risk of infection and severity of Coronavirus Disease-19 (COVID-19). Management of patients infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is challenging due to the scarce scientific information and treatment guidelines. In this work, we present our Institutional experience with our first 100 patients with oncological malignancies and COVID-19. Patients and methods: We conducted a cross-sectional study of the first 100 patients hospitalised at the Instituto Nacional de Enfermedades Neoplasicas (Lima, Peru) who were positive for SARS-CoV-2 by reverse transcriptase (RT)-PCR during the period 30 March to 20 June. Clinicopathological variables of the oncological disease as well as risk factors, management and outcomes to COVID-19 were evaluated. Results: The mean age was 43.5 years old (standard deviations: ±24.8) where 57% were male patients. In total, 44%, 37% and 19% were adult patients bearing solid tumours, adults with haematologic malignancies and paediatric patients, respectively. Hypertension was the most frequent comorbidity (23%) followed by chronic lung disease (10%). COVID-19-associated symptoms included cough (65%), fever (57%) and dyspnoea (56%). Twelve percent of patients were asymptomatic. Nosocomial infections were more frequent in paediatric patients (84.2%) than in adult patients (16.0%). Patients with uncontrolled oncological disease were most frequent (72%). Anaemia was present in 67% of patients, 68% had lymphopenia, 62% had ferritin value > 500 mcg/L, 85% had elevated lactate dehydrogenase (LDH), 83% D-dimer > 500 ng/mL and 80% C-Reactive Protein > 8 mg/L. The most common complication was acute respiratory failure (42%). Overall fatality rate was 39% where the main cause of mortality was acute respiratory distress syndrome (64.1%). Conclusion: Paediatric patients had better outcomes than adult populations, and a high number of asymptomatic carriers and nosocomial infection, early diagnosis are recommended. Considering oncological treatments 30 days before COVID-19 diagnosis, our data did not reveal an increased mortality. / Revisión por pares