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.

Adaptive evolution of PB1 from influenza A(H1N1)pdm09 virus towards an enhanced fitness.

PB1 influenza virus retain traces of interspecies transmission and adaptation. Previous phylogenetic analyses highlighted mutations L298I, R386K and I517V in PB1 to have putatively ameliorated the A(H1N1)pdm09 adaptation to the human host. This study aimed to evaluate the reversal of these mutations and infer the role of these residues in the virus overall fitness and adaptation. We generate PB1-mutated viruses introducing I298L, K386R and V517I mutations in PB1 and evaluate their phenotypic impact on viral growth and on antigen yield. We observed a decrease in viral growth accompanied by a reduction in hemagglutination titer and neuraminidase activity, in comparison with wt. Our data indicate that the adaptive evolution occurred in the PB1 leads to an improved overall viral fitness; and such biologic advantaged has the potential to be applied to the optimization of influenza vaccine seed prototypes.

Optimization of A(H1N1)pdm09 vaccine seed viruses: The source of PB1 and HA vRNA as a major determinant for antigen yield.

Vaccination prevents and reduces the severity of influenza virus infections. Continuous evolution of influenza hemagglutinin (HA) and neuraminidase (NA) supports the virus to evade pre-existing immunity, which demands vaccines to be reformulated every year. Incorporation of polymerase basic protein 1 (PB1) viral RNA (vRNA) of the same origin of HA and NA vRNA has been observed in previous pandemic viruses and occasionally reported for influenza A vaccine prototype strains of prior seasons. At this point, it remains to be explored whether this phenomenon translates into an improved growth phenotype. In this work, we showed that the HA vRNA of A(H1N1)pdm09 is generally incorporated with the PB1 vRNA of the same origin, establishing the beneficial effect of the presence of PB1 and the pattern of the PB1-HA co-incorporation in the A(H1N1)pdm09 model. We further investigated the putative interplay between PB1 and antigenic proteins regarding the vRNA composition of the progeny and observed that vRNA segregation does not appear to be mainly determined by protein-protein interactions; while vRNA-vRNA interactions can be suggested as the main driving force. Our data also indicate an increase in the hemagglutination capacity and neuraminidase activity due to incorporation of PB1, HA and NA from A(H1N1)pdm09, in comparison with the recombinant virus incorporating only HA and NA from A(H1N1)pdm09 - which have the potential to improve current limitations regarding antigenicity and immunogenicity of influenza vaccines. Further knowledge of the complex vRNA-vRNA interaction network between PB1 and HA will additionally contribute to improve current vaccine formulation, and to gradually optimize the production of A(H1N1)pdm09 reverse genetics vaccine seed virus towards a higher cost-effectiveness.

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.

A rare case of pulmonary lymphangiectasia associated with CHD.

CHD may, at times, occur in the framework of other rare pathologies. These, having similar clinical manifestations, present a diagnostic dilemma for the clinician.The authors present the case of an infant with non-syndromic complete atrioventricular septal defect, whose post-operative period was surprisingly complicated by progressive pulmonary hypertension. Despite intensive care, the infant ultimately died. The diagnosis of unilateral primary pulmonary lymphangiectasia was only possible post mortem.

NS1 protein as a novel anti-influenza target: Map-and-mutate antiviral rationale reveals new putative druggable hot spots with an important role on viral replication.

Influenza NS1 is a promising anti-influenza target, considering its conserved and druggable structure, and key function in influenza replication and pathogenesis. Notwithstanding, target identification and validation, strengthened by experimental data, are lacking. Here, we further explored our previously designed structure-based antiviral rationale directed to highly conserved druggable NS1 regions across a broad spectrum of influenza A viruses. We aimed to identify NS1-mutated viruses exhibiting a reduced growth phenotype and/or an altered cell apoptosis profile. We found that NS1 mutations Y171A, K175A (consensus druggable pocket 1), W102A (consensus druggable pocket 3), Q121A and G184P (multiple consensus druggable pockets) - located at hot spots amenable for pharmacological modulation - significantly impaired A(H1N1)pdm09 virus replication, in vitro. This is the first time that NS1-K175A, -W102A, and -Q121A mutations are characterized. Our map-and-mutate strategy provides the basis to establish the NS1 as a promising target using a rationale with a higher resilience to resistance development.

Post-COVID-19 and the Portuguese national eye care system challenge / Post-COVID-19 y el desafío del sistema nacional portugués de atención oftalmológica

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

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.

Using a PCR-Based Method To Analyze and Model Large, Heterogeneous Populations of DNA.

The study of populations of large size and high diversity is limited by the capability of collecting data. Moreover, for a pool of individuals, each associated with a unique characteristic feature, as the pool size grows, the possible interactions increase exponentially and quickly go beyond the limit of computation and experimental studies. Herein, the design of DNA libraries with various diversity is reported. By using a facile analytical method based on real-time PCR, the diversity of a pool of DNA can be evaluated to allow extraordinarily high heterogenicity (e.g., >1 trillion). It is demonstrated that these DNA libraries can be used to model heterogeneous populations; these libraries exhibit functions such as self-protection, suitability for biased expansion, and the possibility to evolve into amorphous structures. The method has shown the remarkable power of parallel computing with DNA, since it can resemble an analogue computer and be applied in selection-based biotechnology methods, such as DNA-encoded chemical libraries. As a chemical approach to solve problems traditionally for genetic and statistical analysis, the method provides a quick and cost-efficient evaluation of library diversity for intermediate steps through a selection process.

Lessons learned from a prolonged norovirus GII.P16-GII.4 Sydney 2012 variant outbreak in a long-term care facility in Portugal, 2017.

OBJECTIVE: To investigate an outbreak of acute gastroenteritis caused by norovirus (NoV) in a long-term care facility (LTCF) in Portugal to describe and estimate its extent, and we implemented control measures. DESIGN: Outbreak investigation. METHODS: Probable cases were residents or staff members in the LTCF with at least 1 of the following symptoms: (1) diarrhea, (2) vomiting, (3) nausea, and/or (4) abdominal pain between October 31 and December 8, 2017. Confirmed cases were probable cases with positive NoV infection detected by real-time polymerase chain reaction (RT-PCR) and the same genotype in stool specimens. RESULTS: The outbreak was caused by NoV GII.P16-GII.4 Sydney 2012 variant and affected 146 people. The highest illness rates were observed in residents (97 of 335, 29%) and nurses (16 of 83, 19%). All 11 resident wards were affected. Data on cases and their working or living areas suggest that movement between wards facilitated the transmission of NoV, likely from person to person. CONCLUSIONS: The delay in the identification of the causative agent, a lack of restrictions of resident and staff movement between wards, and ineffective initial deep-cleaning procedures resulted an outbreak that continued for >1 month. The outbreak ended only after implementation of strict control measures. Recommendations for controlling future NoV outbreaks in LTCFs include emphasizing the need to control resident's movements and to restrict visitors, timely and effective environmental cleaning and disinfection, leave of absence for ill staff, and encouraging effective hand hygiene.

Management of Asymptomatic Wolff-Parkinson-White Pattern in Young Patients: Has Anything Changed?

The approach to pediatric asymptomatic Wolff-Parkinson-White (WPW) patients is controversial. The objective of this review is to update the last consensus of specialists of the Pediatric and Congenital Electrophysiology Society/Heart Rhythm Society on this subject in order to summarize the most recent evidence on the management of young patients with asymptomatic WPW pattern. A systematic review of the literature published between 2008 and 2018 was performed taking into account the protocol of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) in PubMed (including Cochrane), Embase, and Web of Science. Observational, experimental, and multicentric studies were included. Out of a total of 37 articles selected, 4 were considered eligible. Most studies considered a cutoff age of 8 or greater as recommended in the 2012 consensus. The identification of a shortest pre-excitatory RR interval (SPERRI) ≤ 250 ms seems to be the best predictor for risk stratification. The importance of routine isoprenaline use to improve the sensitivity of the electrophysiological study to identify patients at high risk of sudden death was consensual. Prophylactic ablative therapy has been indicated in asymptomatic children with an accessory pathway (AP) who have a low SPERRI and/or a low effective anterograde period of the AP and/or multiple APs. Despite the evidence found in the most recent studies, more studies are warranted in this setting.

To hit or not to hit: Large-scale sequence analysis and structure characterization of influenza A NS1 unlocks new antiviral target potential.

Influenza NS1 protein is among the most promising novel druggable anti-influenza target, based on its structure; multiple interactions; and global function in influenza replication and pathogenesis. Notwithstanding, drug development guidance based on NS1 structural biology is lacking. Here, we design a promising strategy directed to highly conserved druggable regions as a result of an exhaustive large-scale sequence analysis and structure characterization of NS1 protein across human-infecting influenza A subtypes, over the past 100 years. We have identified 3 druggable pockets and 8 new potential hot spot residues in the NS1 protein, not described before, additionally to other 16 sites previously identified, which represent attractive targets for pharmacological modulation. This study provides a rationale towards structure-function studies of NS1 druggable sites, which have the potential to accelerate the NS1 target validation. This research also contributes to a deeper comprehension and insight into the evolutionary dynamics of influenza A NS1 protein.

Rare cause of cardiac failure in an infant.

Congenital stenosis of the pulmonary veins is a rare condition whose outcome is guarded despite the available treatment options. We report a case of a 6-month-old infant with significant stenosis of all four pulmonary veins.

Specificity from nonspecific interaction: regulation of tumor necrosis factor-α activity by DNA.

As anionic biopolymers, oligonucleotides can have biological functions independent from their roles as the medium for the storage and flow of genetic information. In this paper, we investigated the interaction between DNA and the pro-inflammatory cytokine tumor necrosis factor-α (TNFα). Although various forms of DNA bind to TNFα with low µm dissociation constants, the interaction stabilizes the trimeric form of TNFα and enhances its cytotoxic effect. Based on this mechanism, a photoswitchable TNFα (TNFα-2-nitroveratryloxycarbonyl) has been designed whose sensitivity to DNA-mediated up-regulation of TNFα activity can be tuned by light irradiation. The mechanism described in this study represents a general model to understand the involvement of nonspecific interactions among biomolecules in regulating their biological functions. Because the interaction is not DNA sequence-specific, the resulting effect should be considered for oligonucleotide-based therapeutics in general.

Second generation DNA-encoded dynamic combinatorial chemical libraries.

We present a DNA-encoded chemical library, which allows dynamic selection followed by ligation of the encoding strands. As a chemical approach to mimic the genetic recombination process of adaptive immunity, the technology led to an enhanced enrichment factor and signal-to-noise ratio compared to static libraries.

Peptidyl prolyl cis/trans isomerase activity on the cell surface correlates with extracellular matrix development.

Interactions with the extracellular matrix (ECM) dictate cell fates. However, the complexity of dense ECM network and cell-surface molecules prevent the study of their dynamic interaction at the molecular level on living cells. Here, we focus on peptidyl prolyl cis/trans isomerases (PPIases) to dissect prolyl isomerization from other dynamic events. We reveal the contribution of PPIase on the mechanical properties of various ECM materials and on the dynamic cell-ECM interaction. To avoid complications associated with the existing spectroscopy-based methods such as light scattering, an assay was developed for detecting PPIase activity on living cell surface. This assay allows us to correlate PPIase activity with ECM development, and with the physiological and pathological states of the cells, including the functional properties of cancer cells and immune effector cells.