How to dissect viral infections and their interplay with the host-proteome by immunoaffinity and mass spectrometry: A tutorial

[Display omitted] • Immunohistochemistry with magnetic core nanoparticles to isolate viruses. • The use of MALDI-MS for rapid virus detection is explained in detail. • The use of ESI-MS/MS to pinpoint host-patient crosstalk is explained in detail. • The absolute quantitative MS is explained for large-scale protein quantitation. The capabilities of bioanalytical mass spectrometry to (i) detect and differentiate viruses at the peptide level whilst maintaining high sample throughput and (ii) to provide diagnosis and prognosis for infected patients are presented as a tutorial in this work to aid analytical chemists and physicians to gain insights into the possibilities offered by current high-resolution mass spectrometry technology and bioinformatics. From (i) sampling to sample treatment;(ii) Matrix-Assisted Laser Desorption Ionization- to Electrospray Ionization -based mass spectrometry;and (iii) from clustering to peptide sequencing;a detailed step-by-step guide is provided and exemplified using SARS-CoV-2 Spike Y839 variant and the variant of concern SARS-CoV-2 Alpha (B.1.1.7 lineage), Influenza B, and Influenza A subtypes AH1N1pdm09 and AH3N2. [ FROM AUTHOR]

Exploring new antiviral targets for influenza and COVID-19: Mapping promising hot spots in viral RNA polymerases.

Influenza and COVID-19 are infectious respiratory diseases that represent a major concern to public health with social and economic impact worldwide, for which the available therapeutic options are not satisfactory. The RdRp has a central role in viral replication and thus represents a major target for the development of antiviral approaches. In this study, we focused on Influenza A virus PB1 polymerase protein and the betacoronaviruses nsp12 polymerase protein, considering their functional and structural similarities. We have performed conservation and druggability analysis to map conserved druggable regions, that may have functional or structural importance in these proteins. We disclosed the most promising and new targeting regions for the discovery of new potential polymerase inhibitors. Conserved druggable regions of putative interaction with favipiravir and molnupiravir were also mapped. We have also compared and integrated the current findings with previous research.

"How to dissect viral infections and their interplay with the host-proteome by immunoaffinity and mass spectrometry: A tutorial.”

The capabilities of bioanalytical mass spectrometry to (i) detect and differentiate viruses at the peptide level whilst maintaining high sample throughput and (ii) to provide diagnosis and prognosis for infected patients are presented as a tutorial in this work to aid analytical chemists and physicians to gain insights into the possibilities offered by current high-resolution mass spectrometry technology and bioinformatics. From (i) sampling to sample treatment;(ii) Matrix-Assisted Laser Desorption Ionization- to Electrospray Ionization -based mass spectrometry;and (iii) from clustering to peptide sequencing;a detailed step-by-step guide is provided and exemplified using SARS-CoV-2 Spike Y839 variant and the variant of concern SARS-CoV-2 Alpha (B.1.1.7 lineage), Influenza B, and Influenza A subtypes AH1N1pdm09 and AH3N2.

Enhanced In Vitro Antiviral Activity of Hydroxychloroquine Ionic Liquids against SARS-CoV-2.

The development of effective antiviral drugs against SARS-CoV-2 is urgently needed and a global health priority. In light of the initial data regarding the repurposing of hydroxychloroquine (HCQ) to tackle this coronavirus, herein we present a quantitative synthesis and spectroscopic and thermal characterization of seven HCQ room temperature ionic liquids (HCQ-ILs) obtained by direct protonation of the base with two equivalents of organic sulfonic, sulfuric and carboxylic acids of different polarities. Two non-toxic and hydrophilic HCQ-ILs, in particular, [HCQH2][C1SO3]2 and [HCQH2][GlcCOO]2, decreased the virus-induced cytopathic effect by two-fold in comparison with the original drug, [HCQH2][SO4]. Despite there being no significant differences in viral RNA production between the three compounds, progeny virus production was significantly affected (p < 0.05) by [HCQH2][GlcCOO]2. Overall, the data suggest that the in vitro antiviral activities of the HCQ-ILs are most likely the result of specific intra- and intermolecular interactions and not so much related with their hydrophilic or lipophilic character. This work paves the way for the development of future novel ionic formulations of hydroxychloroquine with enhanced physicochemical properties.

COVID-19: impact on Public Health and hypothesis-driven investigations on genetic susceptibility and severity.

COVID-19 is a new complex multisystem disease caused by the novel coronavirus SARS-CoV-2. In slightly over 2 years, it infected nearly 500 million and killed 6 million human beings worldwide, causing an unprecedented coronavirus pandemic. Currently, the international scientific community is engaged in elucidating the molecular mechanisms of the pathophysiology of SARS-CoV-2 infection as a basis of scientific developments for the future control of COVID-19. Global exome and genome analysis efforts work to define the human genetics of protective immunity to SARS-CoV-2 infection. Here, we review the current knowledge regarding the SARS-CoV-2 infection, the implications of COVID-19 to Public Health and discuss genotype to phenotype association approaches that could be exploited through the selection of candidate genes to identify the genetic determinants of severe COVID-19.

Unlocking COVID therapeutic targets: A structure-based rationale against SARS-CoV-2, SARS-CoV and MERS-CoV Spike.

There are no approved target therapeutics against SARS-CoV-2 or other beta-CoVs. The beta-CoV Spike protein is a promising target considering the critical role in viral infection and pathogenesis and its surface exposed features. We performed a structure-based strategy targeting highly conserved druggable regions resulting from a comprehensive large-scale sequence analysis and structural characterization of Spike domains across SARSr- and MERSr-CoVs. We have disclosed 28 main consensus druggable pockets within the Spike. The RBD and SD1 (S1 subunit); and the CR, HR1 and CH (S2 subunit) represent the most promising conserved druggable regions. Additionally, we have identified 181 new potential hot spot residues for the hSARSr-CoVs and 72 new hot spot residues for the SARSr- and MERSr-CoVs, which have not been described before in the literature. These sites/residues exhibit advantageous structural features for targeted molecular and pharmacological modulation. This study establishes the Spike as a promising anti-CoV target using an approach with a potential higher resilience to resistance development and directed to a broad spectrum of Beta-CoVs, including the new SARS-CoV-2 responsible for COVID-19. This research also provides a structure-based rationale for the design and discovery of chemical inhibitors, antibodies or other therapeutic modalities successfully targeting the Beta-CoV Spike protein.

Post-COVID-19 and the Portuguese national eye care system challenge.

The pandemic of the severe acute respiratory syndrome disease caused by the new coronavirus SARS-CoV-2 (COVID-19), had profound impact in many countries and their health care systems. Regarding Portugal, a suppression strategy with social distancing was adopted, attempting to break the transmission chains, bending the epidemy curve and reducing mortality. These measures seek to prevent an eventual National Health Service over-running, enforcing the suspension of all elective and non-urgent health care. Despite the success in so far, there is a consensus on the need to recover the previous level of health care provision and further enhance it. The Portuguese National Health Service, as a public, universal access, health care system funded by the State proved, in this context, its importance and relevance to the Portuguese population. However, long standing issues, such as the pre pandemic over long waiting lists for hospital ophthalmology attendance, whose determinants are fully identified but still unmet, emerge amplified from this pandemic. The lack of primary eye care in the National Health Service is a significant bottleneck, placing a huge stress on hospital-based care. An exclusive ophthalmologist's center care was over-runned before pandemic and will be even more so. The optometrist's exclusion from differentiated, multisectoral and multidisciplinary eye care teams remains the main hurdle to overcome and insure universal eye care in Portugal. National Health Service highlights the consequences of an overcome model. Universal eye care more than ever demands an evidence-based, integrated approach with primary eye care, in the community, on time and of proximity.

La pandemia del síndrome respiratorio agudo grave causado por el nuevo coronavirus SARS-CoV-2 (COVID-19) ha tenido amplias repercusiones en muchos países y en sus sistemas sanitarios. En Portugal, se ha adoptado una estrategia de contención basada en el distanciamiento social, con la cual se ha intentado cortar las cadenas de transmisión, frenar la curva de la epidemia y reducir la mortalidad. Con estas medidas se trataba de evitar un eventual desbordamiento del Servicio Nacional de Salud y se imponía la suspensión de toda la atención médica programada, que no fuera urgente. A pesar del éxito logrado hasta este momento, existe consenso sobre la necesidad de recuperar el nivel anterior de atención médica y fomentar su mejora. El Servicio Nacional de Salud de Portugal, como sistema sanitario público y de acceso universal, a cargo del Estado, ha demostrado, en este contexto, su importancia y pertinencia para la población portuguesa. Sin embargo, los problemas que acarrea desde hace mucho tiempo, como las largas listas de espera, anteriores a la pandemia, en la asistencia oftalmológica hospitalaria, cuyos factores determinantes están completamente identificados, pero que continúan sin solución, se han visto agravados a resultas de esta pandemia. La falta de atención primaria oftalmológica en el Servicio Nacional de Salud es un importante cuello de botella, que ejerce una enorme presión en la atención hospitalaria. La atención de un centro exclusivamente oftalmológico estaba desbordada antes de la pandemia y lo estará aún más después de esta. La exclusión de los optómetras de los equipos de atención oftalmológica diferenciados, multisectoriales y multidisciplinarios continúa siendo el principal obstáculo que debe superar y asegurar la atención oftalmológica universal en Portugal. El Servicio Nacional de Salud hace hincapié en las consecuencias de un modelo superado. La atención oftalmológica universal exige, más que nunca, un enfoque integral basado en la evidencia para abordar la atención primaria oftalmológica en la comunidad, puntual y de proximidad.