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.

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.

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.

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].

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.

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.

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.

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.

The 1918-1919 Influenza Pandemic in Portugal: A Regional Analysis of Death Impact.

Although the impact of deaths occurring during the 1918-1919 influenza pandemic has been assessed in many archeo-epidemiologic studies, detailed estimates are not available for Portugal. We applied negative binomial models to monthly data on respiratory-related and all-cause deaths at the national and district levels from Portugal for 1916-1922. Influenza-related excess mortality was computed as the difference between observed and expected deaths.

Molecular footprints of selective pressure in the neuraminidase gene of currently circulating human influenza subtypes and lineages.

Influenza neuraminidase (NA) is under selective pressure (SP) of both host immune system and drug use. Here, we assembled large datasets of NA sequences of worldwide circulating viruses to estimate the global and site-specific SP acting on all current subtypes/lineages of human influenza NA. An overall negative SP of similar magnitude and a prevalence of negatively selected sites were observed for all subtypes/lineages.

Revision of clinical case definitions: influenza-like illness and severe acute respiratory infection.

The formulation of accurate clinical case definitions is an integral part of an effective process of public health surveillance. Although such definitions should, ideally, be based on a standardized and fixed collection of defining criteria, they often require revision to reflect new knowledge of the condition involved and improvements in diagnostic testing. Optimal case definitions also need to have a balance of sensitivity and specificity that reflects their intended use.

Population genetics of IFITM3 in Portugal and Central Africa reveals a potential modifier of influenza severity.

Influenza epidemics are a serious global public health and economic problem. The IFITM3 allele (rs12252-C) was suggested as a population-based genetic risk factor for severe influenza virus infection by A(H1N1)pdm09. We analyzed the population genetics of IFITM3 variants in the Portuguese general population (n = 200) and Central Africans (largely Angolan) (n = 148) as well as its association to influenza severity in Portuguese patients (n = 41).

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2015-2016.

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) assessed antiviral susceptibility of 14,330 influenza A and B viruses collected by WHO-recognized National Influenza Centres (NICs) between May 2015 and May 2016. Neuraminidase (NA) inhibition assay was used to determine 50% inhibitory concentration (IC) data for NA inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Furthermore, NA sequences from 13,484 influenza viruses were retrieved from public sequence databases and screened for amino acid substitutions (AAS) associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NAIs.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014-2015.

The World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza (WHO CCs) tested 13,312 viruses collected by WHO recognized National Influenza Centres between May 2014 and May 2015 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Ninety-four per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.

Reverse genetics vaccine seeds for influenza: Proof of concept in the source of PB1 as a determinant factor in virus growth and antigen yield.

Growth deficits of reverse genetics vaccine seeds have compromised effective immunization. The impairment has been attributed to sub-optimal protein interactions. Some level of dependence may exist between PB1 and antigenic glycoproteins, however, further research is necessary to clarify the extent to which it can be used in favor of seed production.

[The Museu da Saúde in Portugal: a physical space, a virtual space].

Museu da Saúde (Museum of Health) in Portugal, based on the dual concept of a multifaceted physical space and a virtual space, is preparing an inventory of its archive. So far, it has studied five of its collections in greater depth: tuberculosis, urology, psychology, medicine, and malaria. In this article, these collections are presented, and the specificities of developing museological activities within a national laboratory, Instituto Nacional de Saúde Doutor Ricardo Jorge, are also discussed, highlighting the issues of the store rooms and exhibition spaces, the inventory process, and the communication activities, with a view to overcoming the challenges inherent to operating in a non-museological space.

Distinct kinetics and pathways of apoptosis in influenza A and B virus infection.

Annual influenza epidemics are associated with high incidence and mortality rates, and are an important cause of work absenteeism and productivity losses. For successful replication, influenza viruses have evolved as to counteract and/or take a part on host defense mechanisms. Manipulation of apoptosis is one of such mechanisms that have been subject of attention, particularly in relation to influenza type A viruses over the past years.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014.

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 10,641 viruses collected by WHO-recognized National Influenza Centres between May 2013 and May 2014 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. In addition, neuraminidase (NA) sequence data, available from the WHO CCs and from sequence databases (n=3206), were screened for amino acid substitutions associated with reduced NAI susceptibility. Ninety-five per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe.

Genetic evolution of PB1 in the zoonotic transmission of influenza A(H1) virus.

The epidemiology of human infection with swine-origin influenza A(H1) viruses suggests that the virus must adapt to replicate and transmit within the human host. PB1 is essential to the replication process. The objective of this study was to identify whether PB1 retains genetic traces of interspecies transmission and adaptation.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2012-2013.

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir.

Influenza A(H1N1)pdm09 resistance and cross-decreased susceptibility to oseltamivir and zanamivir antiviral drugs.

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011.

Guidance for clinical and public health laboratories testing for influenza virus antiviral drug susceptibility in Europe.

Two classes of antiviral drugs are licensed in Europe for treatment and prophylaxis of influenza; the M2 ion-channel blockers amantadine and rimantadine acting against type A influenza viruses only and the neuraminidase enzyme inhibitors zanamivir and oseltamivir acting against type A and type B influenza viruses. This guidance document was developed for but not limited to the European Union (EU) and other European Economic Area (EEA) countries on how and when to test for influenza virus antiviral drug susceptibility. It is aimed at clinical and influenza surveillance laboratories carrying out antiviral drug susceptibility testing on influenza viruses from patients suspected of harbouring viruses with reduced susceptibility or for the monitoring of the emergence of such among circulating viruses, respectively.

Genomic signatures and antiviral drug susceptibility profile of A(H1N1)pdm09.

Background: Genetic changes in influenza surface and internal genes can alter viral fitness and virulence. Mutation trend analysis and antiviral drug susceptibility profiling of A(H1N1)pdm09 viruses is essential for risk assessment of emergent strains and disease management.

Objective: To profile genomic signatures and antiviral drug resistance of A(H1N1)pdm09 viruses and to discuss the potential role of mutated residues in human host adaptation and virulence.

Antibody response to the influenza vaccine in healthcare workers.

People vaccinated against influenza develop hemagglutination inhibition (HAI) antibodies (Ab) that bind to the virus and neutralize it. Ab titer levels are variable depending on factors insufficiently studied, and tend to decrease over time. In the present study, we analyzed antibodies responses before and after influenza vaccination in nurses working in a hospital, with the aim of: - identifying seroconversion rates to trivalent influenza vaccine one month after immunization; - evaluating if, six months after vaccination, influenza HAI Ab titer fall comparing to one-month post vaccination HAI Ab titer; - studying the association between the lack of HAI Ab response (seroconversion) assessed one month after immunization and: ◦ past influenza vaccinations, ◦ baseline (before vaccination) HAI Ab titer, ◦ baseline (before vaccination) HAI Ab titer ≥ 40 (considered as a protection titer).

Excess mortality associated with influenza epidemics in Portugal, 1980 to 2004.

Background: Influenza epidemics have a substantial impact on human health, by increasing the mortality from pneumonia and influenza, respiratory and circulatory diseases, and all causes. This paper provides estimates of excess mortality rates associated with influenza virus circulation for 7 causes of death and 8 age groups in Portugal during the period of 1980-2004.

Methodology/principal Findings: We compiled monthly mortality time series data by age for all-cause mortality, cerebrovascular diseases, ischemic heart diseases, diseases of the respiratory system, chronic respiratory diseases, pneumonia and influenza.