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The response to COVID-19 altered global dispersal of influenza viruses.

Tens of thousands of influenza sequences are deposited into the GenBank database each year. The software tool FLu ANnotation tool (FLAN) has been used by GenBank since 2007 to validate and annotate incoming influenza sequence submissions and has been publicly available as a webserver but not as a standalone tool. Viral Annotation DefineR (VADR) is a general sequence validation and annotation software package used by GenBank for norovirus, dengue virus and SARS-CoV-2 virus sequence processing that is available as a standalone tool. We have created VADR influenza models based on the FLAN reference sequences and adapted VADR to accurately annotate influenza sequences. VADR and FLAN show consistent results on the vast majority of influenza sequences, and when they disagree, VADR is usually correct. VADR can also accurately process influenza D sequences as well as influenza A H17, H18, H19, N10 and N11 subtype sequences, which FLAN cannot. VADR 1.6.3 and the associated influenza models are now freely available for users to download and use. Database URL: https://bitbucket.org/nawrockie/vadr-models-flu., (Published by Oxford University Press 2024. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Purified subunit viral antigens are weakly immunogenic and stimulate only the antibody but not the T cell-mediated immune response. An alternative approach to inducing protective immunity with small viral peptides may be the targeting of viral epitopes to immunocompetent cells by DNA and protein-engineered vaccines. This review will focus on DNA and protein-generated chimeric molecules carrying engineered fragments specific for activating cell surface co-receptors for inducing protective antiviral immunity. Adjuvanted protein-based vaccine or DNA constructs encoding simultaneously T - and B-cell peptide epitopes from influenza viral hemagglutinin, and scFvs specific for costimulatory immune cell receptors may induce a significant increase of anti-influenza antibody levels and strong CTL activity against virus-infected cells in a manner that mimics the natural infection. Here we summarize the development of several DNA and protein chimeric constructs carrying influenza virus HA317-41 fragment. The generated engineered molecules were used for immunization in intact murine and experimentally humanized NSG mouse models.

Recent studies have documented prolonged expression of viral antigens and RNA and associated inflammation after infection with SARS-CoV-2 in a substantial proportion of infected patients. The persisting SARS-CoV-2 effects and findings, with inflammation associated with continued detection of viral antigens, especially resemble those previously reported for influenza virus, as well as respiratory syncytial virus (RSV). The reports indicate the need for improved insight into the mechanisms whereby post-SARS-CoV-2 infection-related illness is apparently more common and perhaps even more persistent after infection than observed for other respiratory viruses.

Here, we report the results of a monitoring study of bat viruses in Austria to strengthen the knowledge of circulating viruses in Austrian bat populations. In this study, we analyzed 618 oropharyngeal and rectal swab samples from 309 bats and 155 pooled tissue samples from dead bats. Samples were collected from 18 different bat species from multiple locations in Austria, from November 2015 to April 2018, and examined for astroviruses, bornaviruses, coronaviruses, hantaviruses, morbilliviruses, orthomyxoviruses (influenza A/C/D viruses), pestiviruses and rhabdoviruses (lyssaviruses) using molecular techniques and sequencing. Using RT-qPCR, 36 samples revealed positive or suspicious results for astroviruses, Brno-hantaviruses, and coronaviruses in nine different bat species. Further sequencing revealed correspondent sequences in five samples. In contrast, none of the tested samples was positive for influenza viruses A/C/D, bornaviruses, morbilliviruses, lyssaviruses, or pestiviruses.

Influenza viruses rapidly evolve to evade previously acquired human immunity. Maintaining vaccine efficacy necessitates continuous monitoring of antigenic differences among strains. Traditional serological methods for assessing these differences are labor-intensive and time-consuming, highlighting the need for efficient computational approaches. This paper proposes MetaFluAD, a meta-learning-based method designed to predict quantitative antigenic distances among strains. This method models antigenic relationships between strains, represented by their hemagglutinin (HA) sequences, as a weighted attributed network. Employing a graph neural network (GNN)-based encoder combined with a robust meta-learning framework, MetaFluAD learns comprehensive strain representations within a unified space encompassing both antigenic and genetic features. Furthermore, the meta-learning framework enables knowledge transfer across different influenza subtypes, allowing MetaFluAD to achieve remarkable performance with limited data. MetaFluAD demonstrates excellent performance and overall robustness across various influenza subtypes, including A/H3N2, A/H1N1, A/H5N1, B/Victoria, and B/Yamagata. MetaFluAD synthesizes the strengths of GNN-based encoding and meta-learning to offer a promising approach for accurate antigenic distance prediction. Additionally, MetaFluAD can effectively identify dominant antigenic clusters within seasonal influenza viruses, aiding in the development of effective vaccines and efficient monitoring of viral evolution., (© The Author(s) 2024. Published by Oxford University Press.)

Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy., (© 2024. The Author(s).)

In this study, we investigated the concentration of airborne influenza virus in daycare centers and influencing factors, such as common cold prevalence, air pollutants, and meteorological factors. A total of 209 air samples were collected from daycare centers in Kaohsiung and the influenza virus was analyzed using real-time quantitative polymerase chain reaction. Air pollutants and metrological factors were measured using real-time monitoring equipment. Winter had the highest positive rates of airborne influenza virus and the highest prevalence of the common cold, followed by summer and autumn. The concentration of CO was significantly positively correlated with airborne influenza virus. Daycare center A, with natural ventilation and air condition systems, had a higher concentration of airborne influenza A virus, airborne fungi, and airborne bacteria, as well as a higher prevalence of the common cold, than daycare center B, with a mechanical ventilation system and air purifiers, while the concentrations of CO 2 , CO, and UFPs in daycare center A were lower than those in daycare center B. We successfully detected airborne influenza virus in daycare centers, demonstrating that aerosol sampling for influenza can provide novel epidemiological insights and inform the management of influenza in daycare centers.

The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co-expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin-related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are both respiratory viruses with similar clinical manifestations and modes of transmission. This study describes influenza data before and during the coronavirus disease pandemic (COVID-19) in Cameroon and SARS-CoV-2 data during the pandemic period., Methods: The study ran from 2017 to 2022, and data were divided into two periods: before (2017-2019) and during (2020-2022) the COVID-19 pandemic. Nasopharyngeal samples collected from persons with respiratory illness were tested for influenza using the Centers for Disease Control and Prevention (CDC) typing and subtyping assays. During the COVID-19 pandemic, the respiratory specimens were simultaneously tested for SARS-CoV-2 using the DaAn gene protocol or the Abbott real-time SARS-CoV-2 assay. The WHO average curve method was used to compare influenza virus seasonality before and during the pandemic., Results: A total of 6246 samples were tested. Influenza virus detection rates were significantly higher in the pre-pandemic period compared to the pandemic period (30.8% vs. 15.5%; p < 0.001). Meanwhile, the SARS-CoV-2 detection rate was 2.5%. A change in the seasonality of influenza viruses was observed from a bi-annual peak before the pandemic to no clear seasonal pattern during the pandemic. The age groups 2-4 and 5-14 years were significantly associated with higher influenza positivity rates in both pre-pandemic and pandemic periods. For SARS-CoV-2, all age groups above 15 years were the most affected population., Conclusion: The COVID-19 pandemic had a significant impact on the seasonal influenza by changing the seasonality of the virus and reducing its detection rates., (© 2024 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Background: Novel influenza viruses pose a potential pandemic risk, and rapid detection of infections in humans is critical to characterizing the virus and facilitating the implementation of public health response measures., Methods: We use a probabilistic framework to estimate the likelihood that novel influenza virus cases would be detected through testing in different community and healthcare settings (urgent care, emergency department, hospital, and intensive care unit [ICU]) while at low frequencies in the United States. Parameters were informed by data on seasonal influenza virus activity and existing testing practices., Results: In a baseline scenario reflecting the presence of 100 novel virus infections with similar severity to seasonal influenza viruses, the median probability of detecting at least one infection per month was highest in urgent care settings (72%) and when community testing was conducted at random among the general population (77%). However, urgent care testing was over 15 times more efficient (estimated as the number of cases detected per 100,000 tests) due to the larger number of tests required for community testing. In scenarios that assumed increased clinical severity of novel virus infection, median detection probabilities increased across all healthcare settings, particularly in hospitals and ICUs (up to 100%) where testing also became more efficient., Conclusions: Our results suggest that novel influenza virus circulation is likely to be detected through existing healthcare surveillance, with the most efficient testing setting impacted by the disease severity profile. These analyses can help inform future testing strategies to maximize the likelihood of novel influenza detection., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Background: Although influenza viruses cause only one-fifth of severe acute respiratory infections (SARI) in Burkina Faso, the other viral causes of SARI remain poorly investigated to inform clinical and preventive decision making., Methods: Between 2016 and 2019, we prospectively enrolled inpatients meeting the World Health Organization (WHO) case definition of SARI in Burkina Faso. Results of viral etiologies among inpatients tested negative for influenza using the Fast Track Diagnostics Respiratory Kits (FTD-33) were reported., Results: Of 1541 specimens tested, at least one respiratory virus was detected in 76.1% of the 1231 specimens negative for influenza virus. Human rhinoviruses (hRVs) were the most detected pathogens (476; 38.7%), followed by human adenoviruses (hAdV) (17.1%, 210/1231), human respiratory syncytial virus (hRSV) (15.4%, 189/1231), enterovirus (EnV) (11.2%, 138/1231), human bocavirus (hBoV) (7.9%, 97/1231), parainfluenza 3 (hPIV3) (6.1%, 75/1231), human metapneumovirus (hMPV) (6.0%,74/1321), parainfluenza 4 (hPIV4) (4.1%, 51/1231), human coronavirus OC43 (hCoV-OC43) (3.4%, 42/1231), human coronavirus HKU1(hCoV-HKU1) (2.7%, 33/1231), human coronavirus NL63 (hCoV-NL63) (2.5%, 31/1231), parainfluenza 1 (hPIV1) (2.0%, 25/1231), parainfluenza 2 (hPIV2) (1.8%, 22/1231), human parechovirus (PeV) (1.1%, 14/1231), and human coronavirus 229E (hCoV-229E) (0.9%, 11/1231). Among SARI cases, infants aged 1-4 years were mostly affected (50.7%; 622/1231), followed by those <1 year of age (35.7%; 438/1231). Most detected pathogens had year-long circulation patterns, with seasonal peaks mainly observed during the cold and dry seasons., Conclusion: Several non-influenza viruses are cause of SARI in Burkina Faso. The integration of the most common pathogens into the routine influenza surveillance system might be beneficial., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities., (© 2024. The Author(s).)

Genetic reassortment of avian, swine, and human influenza A viruses (IAVs) poses potential pandemic risks. Surveillance is important for influenza pandemic preparedness, but the susceptibility of zoonotic IAVs to the cap-dependent endonuclease inhibitor baloxavir acid (BXA) has not been thoroughly researched. Although an amino acid substitution at position 38 in the polymerase acidic protein (PA/I38) in seasonal IAVs reduces BXA susceptibility, PA polymorphisms at position 38 are rarely seen in zoonotic IAVs. Here, we examined the impact of PA/I38 substitutions on the BXA susceptibility of recombinant A(H5N1) viruses. PA mutants that harbored I38T, F, and M were 48.2-, 24.0-, and 15.5-fold less susceptible, respectively, to BXA than wild-type A(H5N1) but were susceptible to the neuraminidase inhibitor oseltamivir acid and the RNA polymerase inhibitor favipiravir. PA mutants exhibited significantly impaired replicative fitness in Madin-Darby canine kidney cells at 24 h postinfection. In addition, in order to investigate new genetic markers for BXA susceptibility, we screened geographically and temporally distinct IAVs isolated worldwide from birds and pigs. The results showed that BXA exhibited antiviral activity against avian and swine viruses with similar levels to seasonal isolates. All viruses tested in the study lacked the PA/I38 substitution and were susceptible to BXA. Isolates harboring amino acid polymorphisms at positions 20, 24, and 37, which have been implicated in the binding of BXA to the PA endonuclease domain, were also susceptible to BXA. These results suggest that monitoring of the PA/I38 substitution in animal-derived influenza viruses is important for preparedness against zoonotic influenza virus outbreaks., (© 2024. The Author(s).)

Influenza viruses undergo frequent genomic mutations, leading to potential cross-species transmission, phenotypic changes, and challenges in diagnostic reagents and vaccines. Accurately evaluating and predicting the risk of such variations remain significant challenges. To address this, we developed the VarEPS-Influ database, an influenza virus variations risk evaluation system (VarEPS-Influ). This database employs a 'multi-dimensional evaluation of mutations' strategy, utilizing various tools to assess the physical and chemical properties, primary, secondary, and tertiary structures, receptor affinity, antibody binding capacity, antigen epitopes, and other aspects of the variation's impact. Additionally, we consider space-time distribution, host species distribution, pedigree analysis, and frequency of mutations to provide a comprehensive risk evaluation of mutations and viruses. The VarEPS-Influ database evaluates both observed variations and virtual variations (variations that have not yet occurred), thereby addressing the time-lag issue in risk predictions. Our current one-stop evaluation system for influenza virus genomic variation integrates 1065290 sequences from 224 927 Influenza A, B and C isolates retrieved from public resources. Researchers can freely access the data at https://nmdc.cn/influvar/., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Viruses in the thogotovirus genus of the family Orthomyxoviridae are much less well-understood than influenza viruses despite documented zoonotic transmission and association with human disease. This study therefore developed a cell-cell fusion assay and three pseudotyping tools and used them to assess envelope function and cell tropism. Envelope glycoproteins of Dhori (DHOV), Thogoto (THOV), Bourbon, and Sinu viruses were all revealed to exhibit pH-dependent triggering of membrane fusion. Lentivirus vectors were robustly pseudotyped with these glycoproteins while influenza virus vectors showed pseudotyping compatibility, albeit at lower efficiencies. Replication-competent vesicular stomatitis virus expressing DHOV or THOV glycoproteins were also successfully generated. These pseudotyped viruses mediated entry into a wide range of mammalian cell lines, including human primary cells. The promiscuousness of these viruses suggests the use of a relatively ubiquitous receptor and their entry into numerous mammalian cells emphasize their high potential as veterinary and zoonotic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier., Objective: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method., Materials and Methods: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct., Results: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase., Conclusion: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

Influenza D virus (IDV) can infect various livestock animals, such as cattle, swine, and small ruminants, and was shown to have zoonotic potential. Therefore, it is important to identify viral factors involved in the broad host tropism and identify potential antiviral compounds that can inhibit IDV infection. Recombinant reporter viruses provide powerful tools for studying viral infections and antiviral drug discovery. Here we present the generation of a fluorescent reporter IDV using our previously established reverse genetic system for IDV. The mNeonGreen (mNG) fluorescent reporter gene was incorporated into the IDV non-structural gene segment as a fusion protein with the viral NS1 or NS2 proteins, or as a separate protein flanked by two autoproteolytic cleavage sites. We demonstrate that only recombinant reporter viruses expressing mNG as an additional separate protein or as an N-terminal fusion protein with NS1 could be rescued, albeit attenuated, compared to the parental reverse genetic clone. Serial passaging experiments demonstrated that the mNG gene is stably integrated for up to three passages, after which internal deletions accumulate. We conducted a proof-of-principle antiviral screening with the established fluorescent reporter viruses and identified two compounds influencing IDV infection. These results demonstrate that the newly established recombinant IDV reporter virus can be applied for antiviral drug discovery and monitoring viral replication, adding a new molecular tool for investigating IDV.

Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ends and encode proteins with no known homologues, apart from the segment 1, which encodes an orthomyxo-like RNA-dependent-RNA polymerase core subunit. Here we show that segments 1-3 encode respectively the PB1, PB2 and PA-like subunits of an active heterotrimeric polymerase that maintains all domains found in the distantly related influenza polymerase, despite an unprecedented overall size reduction of 40%. Multiple high-resolution cryo-EM structures of TiLV polymerase in pre-initiation, initiation and active elongation states, show how it binds the vRNA and cRNA promoters and performs RNA synthesis, with both transcriptase and replicase configurations being characterised. However, the highly truncated endonuclease-like domain appears inactive and the putative cap-binding domain is autoinhibited, emphasising that many functional aspects of TiLV polymerase remain to be elucidated., (© 2023. The Author(s).)

Seasonal influenza viruses cause recurring global epidemics by continually evolving to escape host immunity. The viral constraints and host immune responses that limit and drive the evolution of these viruses are increasingly well understood. However, it remains unclear how most of these advances improve the capacity to reduce the impact of seasonal influenza viruses on human health. In this Review, we synthesize recent progress made in understanding the interplay between the evolution of immunity induced by previous infections or vaccination and the evolution of seasonal influenza viruses driven by the heterogeneous accumulation of antibody-mediated immunity in humans. We discuss the functional constraints that limit the evolution of the viruses, the within-host evolutionary processes that drive the emergence of new virus variants, as well as current and prospective options for influenza virus control, including the viral and immunological barriers that must be overcome to improve the effectiveness of vaccines and antiviral drugs., (© 2023. Springer Nature Limited.)

The emergence of previously unknown disease-causing viruses in mammals is in part the result of a long-term evolutionary process. Reconstructing the deep phylogenetic histories of viruses helps identify major evolutionary transitions and contextualizes the emergence of viruses in new hosts. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the RNA virus order Articulavirales , which includes the influenza viruses. We identified instances of Articulavirales in the invertebrate phylum Cnidaria (including corals), constituting a novel and divergent family that we provisionally named the "Cnidenomoviridae." We further extended the evolutionary history of the influenza virus lineage by identifying four divergent, fish-associated influenza-like viruses, thereby supporting the hypothesis that fish were among the first hosts of influenza viruses. In addition, we substantially expanded the phylogenetic diversity of quaranjaviruses and proposed that this genus be reclassified as a family-the "Quaranjaviridae." Within this putative family, we identified a novel arachnid-infecting genus, provisionally named "Cheliceravirus." Notably, we observed a close phylogenetic relationship between the Crustacea- and Chelicerata-infecting "Quaranjaviridae" that is inconsistent with virus-host codivergence. Together, these data suggest that the Articulavirales has evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of the Articulavirales was likely shaped by multiple aquatic-terrestrial transitions and substantial host jumps, some of which are still observable today.

Importance: The number of known virus species has increased dramatically through metagenomic studies, which search genetic material sampled from a host for non-host genes. Here, we focus on an important viral family that includes influenza viruses, the Orthomyxoviridae , with over 100 recently discovered viruses infecting hosts from humans to fish. We find that one virus called Wǔhàn mosquito virus 6, discovered in mosquitoes in China, has spread across the globe very recently. Surface proteins used to enter cells show signs of rapid evolution in Wǔhàn mosquito virus 6 and its relatives which suggests an ability to infect vertebrate animals. We compute the rate at which new orthomyxovirus species discovered add evolutionary history to the tree of life, predict that many viruses remain to be discovered, and discuss what appropriately designed future studies can teach us about how diseases cross between continents and species., Competing Interests: The authors declare no conflict of interest.

Background: Bacterial and viral infections are commonly implicated in the development of pneumonia. We aimed to compare the diversity and composition of lung bacteria among severe pneumonia patients who were influenza virus positive (IFVP) and influenza virus negative (IFVN)., Methods: Bronchoalveolar lavage fluid specimens were procured from patients diagnosed with severe pneumonia to investigate the microbiome utilizing 16S-rDNA sequencing. The alpha diversity of the microbiome was evaluated employing Chao1, Shannon, and Simpson indexes, while the beta diversity was assessed using principal component analysis and principal coordinate analysis. Linear discriminant analysis effect size (LEfSe) was employed to determine the taxonomic differences between the IFVP and IFVN groups., Results: A total of 84 patients with 42 in the IFVP group and 42 in the IFVN group were enrolled. Slightly higher indexes of Shannon and Simpson were observed in the IFVP group without statistically significant difference. The dominant bacterial genera were Streptococcus, Klebsiella, Escherichia-Shigella in the IFVN group and Acinetobacter, Streptococcus, Staphylococcus in the IFVP group. Streptococcus pneumoniae and Acinetobacter baumannii were the most abundant species in the IFVN and IFVP groups, respectively. LEfSe analysis indicated a greater abundance of Klebsiella in the IFVN group., Conclusions: Individuals with severe pneumonia infected with IFV exhibit heightened susceptibility to certain bacteria, especially Acinetobacter baumannii, and the underlying mechanism of the interaction between IFV and Acinetobacter baumannii in the progression of pneumonia needs further investigation., (© 2023. The Author(s).)

1. Myxovirus resistance (Mx) is a protein produced by the interferon-induced natural immune response with broad spectrum antiviral function. However, the role and expression characteristics of the Mx gene in immune defence against viral infection in goose have not yet been reported.2. This study found a 2576 bp genomic sequence and a 2112 bp mRNA sequence for Mx , encoding 703 amino acids. Multiple sequence alignments of the amino acid sequences showed that the Yangzhou goose Mx (goMx) had 86.99% similarity to the mallard duck ( Ana s platyrhynchos ).3. Tissue-specific expression profiling revealed that the expression of goMx was highest in the lung and spleen. Both poly (I:C) and GPV were found to elevate the expression of goMx . The upregulated expression of goMx was associated with interferon pathway-related genes IRF7 , JAK1 , STAT1 , and STAT2 . Furthermore, overexpression of goMx significantly activated the transcription of poly (I:C) induced TNF-α , IL-1β , IL-6 , and IL-18 .4. The findings of this study suggest that the goMx modulation of the antiviral response is mediated by the interferon pathway.

Influenza viruses pose great threats to public health and cause enormous economic losses every year. Previous work has revealed the viral factors associated with the virulence of influenza viruses in mammals. However, taking prior viral knowledge represented by heterogeneous categorical and discrete information into account to explore virus virulence is scarce in the existing work. How to make full use of the preceding domain knowledge in virulence study is challenging but beneficial. This paper proposes a general framework named ViPal for virulence prediction in mice that incorporates discrete prior viral mutation and reassortment information based on all eight influenza segments. The posterior regularization technique is leveraged to transform prior viral knowledge into constraint features and integrated into the machine learning models. Experimental results on influenza genomic datasets validate that our proposed framework can improve virulence prediction performance over baselines. The comparison between ViPal and other existing methods shows the computational efficiency of our framework with comparable or superior performance. Moreover, the interpretable analysis through SHAP (SHapley Additive exPlanations) identifies the scores of constraint features contributing to the prediction. We hope this framework could provide assistance for the accurate detection of influenza virulence and facilitate flu surveillance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The entry of influenza virus into the host cell requires fusion of its lipid envelope with the host membrane. It is catalysed by viral hemagglutinin protein, whose fragments called fusion peptides become inserted into the target bilayer and initiate its merging with the viral membrane. Isolated fusion peptides are already capable of inducing lipid mixing between liposomes. Years of studies indicate that upon membrane binding they form bend helical structure whose degree of opening fluctuates between tightly closed hairpin and an extended boomerang. The actual way in which they initiate fusion remains elusive. In this work we employ atomistic simulations of wild type and fusion inactive W14A mutant of influenza fusion peptides confined between two closely apposed lipid bilayers. We characterise peptide induced membrane perturbation and determine the potential of mean force for the formation of the first fusion intermediate, an interbilayer lipid bridge called stalk. Our results demonstrate two routes through which the peptides can lower free energy barrier towards fusion. The first one assumes peptides capability to adopt transmembrane configuration which subsequently promotes the creation of a stalk-hole complex. The second involves surface bound peptide configuration and proceeds owing to its ability to stabilise stalk by fitting into the region of extreme negative membrane curvature resulting from its formation. In both cases, the active peptide conformation corresponds to tight helical hairpin, whereas extended boomerang geometry appears to be unable to provide favourable thermodynamic effect. The latter observation offers plausible explanation for long known inactivity of boomerang-stabilising W14A mutation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Michalski, Setny. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export., (© 2023. The Author(s).)

Human diseases, particularly infectious diseases and cancers, pose unprecedented challenges to public health security and the global economy. The development and distribution of novel prophylactic and therapeutic vaccines are the prioritized countermeasures of human disease. Among all vaccine platforms, viral vector vaccines offer distinguished advantages and represent prominent choices for pathogens that have hampered control efforts based on conventional vaccine approaches. Currently, viral vector vaccines remain one of the best strategies for induction of robust humoral and cellular immunity against human diseases. Numerous viruses of different families and origins, including vesicular stomatitis virus, rabies virus, parainfluenza virus, measles virus, Newcastle disease virus, influenza virus, adenovirus and poxvirus, are deemed to be prominent viral vectors that differ in structural characteristics, design strategy, antigen presentation capability, immunogenicity and protective efficacy. This review summarized the overall profile of the design strategies, progress in advance and steps taken to address barriers to the deployment of these viral vector vaccines, simultaneously highlighting their potential for mucosal delivery, therapeutic application in cancer as well as other key aspects concerning the rational application of these viral vector vaccines. Appropriate and accurate technological advances in viral vector vaccines would consolidate their position as a leading approach to accelerate breakthroughs in novel vaccines and facilitate a rapid response to public health emergencies., (© 2023. The Author(s).)

An infectious disease emerged in recent years, Tilapia Lake Virus Disease (TiLVD), has severely restricted the development of global tilapia industry. Vaccination has proved potential strategy to prevent its causative agent Tilapia Lake Virus (TiLV) infectious. However, the response intensity of subunit vaccine is limited by its low immunogenicity, thus inclusion of adjuvants is required. Thus, we prepared a biomimetic nano-system (Cs-S2@M-M) with a particle size of ∼100 nm and an encapsulation efficiency of about 79.15% based on erythrocyte membrane. The immune response was detected after intramuscular injection to assess the effectiveness of the vaccine. The biomimetic system significantly up-regulates the expression of immune genes, enhances the activity of non-specific immune-related enzymes (P < 0.05) and improved relative percentage survival by 17.4%-26.1% in TiLV challenge. The biomimetic nano-system based on erythrocyte membrane induced significant immune response in tilapia and enhanced protection against TiLV, promising as a model for fish vaccines., Competing Interests: Declaration of interest statement The authors have no conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Influenza virus contains a single-stranded negative-sense RNA genome. Replication of the genome is carried out by the viral RNA-dependent RNA polymerase in the context of the viral ribonucleoprotein (RNP) complex, through a positive-sense complementary RNA intermediate. Genome replication is tightly controlled through interactions with accessory viral and host factors. Propelled by developments in recombinant protein expression, and technical improvements in X-ray crystallography and cryo-electron microscopy, snapshots of the replication process have been captured. Here, we review how recent structural data shed light on the molecular mechanisms of influenza virus genome replication, in particular, encapsidation of nascent RNA, de novo RNP assembly, and regulation of replication initiation through interactions with host and viral cues., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Reverse transcription polymerase chain reaction (RT-PCR) on respiratory tract swabs has become the gold standard for sensitive and specific detection of influenza virus, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this retrospective analysis, we report on the successive implementation and routine use of multiplex RT-PCR testing for patients admitted to the Internal Medicine Emergency Department (ED) at a tertiary care center in Western Austria, one of the hotspots in the early coronavirus disease 2019 (COVID-19) pandemic in Europe. Our description focuses on the use of the Cepheid ® Xpert ® Xpress closed RT-PCR system in point-of-care testing (POCT). Our indications for RT-PCR testing changed during the observation period: From the cold season 2016/2017 until the cold season 2019/2020, we used RT-PCR to diagnose influenza or RSV infection in patients with fever and/or respiratory symptoms. Starting in March 2020, we used the RT-PCR for SARS-CoV-2 and a multiplex version for the combined detection of all these three respiratory viruses to also screen subjects who did not present with symptoms of infection but needed in-hospital medical treatment for other reasons. Expectedly, the switch to a more liberal RT-PCR test strategy resulted in a substantial increase in the number of tests. Nevertheless, we observed an immediate decline in influenza virus and RSV detections in early 2020 that coincided with public SARS-CoV-2 containment measures. In contrast, the extensive use of the combined RT-PCR test enabled us to monitor the re-emergence of influenza and RSV detections, including asymptomatic cases, at the end of 2022 when COVID-19 containment measures were no longer in place. Our analysis of PCR results for respiratory viruses from a real-life setting at an ED provides valuable information on the epidemiology of those infections over several years, their contribution to morbidity and need for hospital admission, the risk for nosocomial introduction of such infection into hospitals from asymptomatic carriers, and guidance as to how general precautions and prophylactic strategies affect the dynamics of those infections.

The prevalence of coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus among referred patients in Hamadan province, Iran, from November 2, 2021, to January 30, 2022, was evaluated. Samples were obtained from 14,116 individuals with COVID-19 symptoms and screened for SARS-CoV-2 and influenza viruses using a multiplex real-time PCR panel assay. Of these patients, 14.19%, 17.11%, and 1.35% were infected with influenza virus, SARS-CoV-2, and both viruses, respectively. The majority of the coinfected patients were female outpatients aged 19-60 years., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Defective interfering particles (DIPs) are particles containing defective viral genomes (DVGs) generated during viral replication. DIPs have been found in various RNA viruses, especially in influenza viruses. Evidence indicates that DIPs interfere with the replication and encapsulation of wild-type viruses, namely standard viruses (STVs) that contain full-length viral genomes. DIPs may also activate the innate immune response by stimulating interferon synthesis. In this review, the underlying generation mechanisms and characteristics of influenza virus DIPs are summarized. We also discuss the potential impact of DIPs on the immunogenicity of live attenuated influenza vaccines (LAIVs) and development of influenza vaccines based on NS1 gene-defective DIPs. Finally, we review the antiviral strategies based on influenza virus DIPs that have been used against both influenza virus and SARS-CoV-2. This review provides systematic insights into the theory and application of influenza virus DIPs.

The global outbreak of coronavirus disease 2019 (COVID-19), an emerging disease caused by severe acute respiratory syndrome virus-2 (SARS-CoV-2), and strict restrictions implemented to control the infection have impacted the circulation and transmission of common seasonal viruses worldwide and subsequently the rate of hospitalizations in children at young ages. Respiratory syncytial virus (RSV) surprisingly disappeared in 2020-2021 in many countries due to lockdown and precautions were taken because of the COVID-19 pandemic. Herein, we showed a notable change in the rate of hospitalization and reported an unpredictable outbreak of RSV in a small proportion of children admitted to a children's hospital in Dezful (a city in Southwest Iran) in the early spring of 2022. We performed a descriptive study of hospitalized young children (aged ≤ 5 years) with acute respiratory infections. Together with clinical information, 30 nasopharyngeal swabs were prospectively collected and 3 important respiratory viruses (RSV, influenza viruses, and SARS-CoV-2) were tested through the real-time polymerase chain reaction (real-time PCR) method. The age distribution of 30 hospital-admitted children was 1 month to 5 years old and males were the most included subjects 18/30 (60%) in this study. Although the viral genome of SARS-CoV-2 and influenza viruses was not detected, the presence of RSV was confirmed in 16/30 (53.33%) patients. Results showed that the majority of RSV-infected cases were males 10/16 (62.5%), within 12 months of life, and had changes in parameters of the complete blood count. Almost all patients with RSV infection had a cough as the most common clinical manifestation and had no history of past medical conditions as a risk factor. The presented study is the first investigation that documented an outbreak of RSV infection in young children reported since the onset of the COVID-19 outbreak in Iran. Our cases highlight the potential threats of important but neglected pathogens during the ongoing pandemic as described here for RSV, which would be challenging by easing the preimposed restrictions., (© 2022 Wiley Periodicals LLC.)

Dependent on the excretion pattern, wastewater monitoring of viruses can be a valuable approach to characterizing their circulation in the human population. Using polyethylene glycol precipitation and reverse transcription-quantitative PCR, the occurrence of RNA of SARS-CoV-2 and influenza viruses A/B in the raw wastewater of two treatment plants in Germany between January and May 2022 was investigated. Due to the relatively high incidence in both exposal areas (plant 1 and plant 2), SARS-CoV-2-specific RNA was determined in all 273 composite samples analyzed (concentration of E gene: 1.3 × 10 4 to 3.2 × 10 6 gc/L). Despite a nation-wide low number of confirmed infections, influenza virus A was demonstrated in 5.2% (concentration: 9.8 × 10 2 to 8.4 × 10 4 gc/L; plant 1) and in 41.6% (3.6 × 10 3 to 3.0 × 10 5 gc/L; plant 2) of samples. Influenza virus B was detected in 36.0% (7.2 × 10 2 to 8.5 × 10 6 gc/L; plant 1) and 57.7% (9.6 × 10 3 to 2.1 × 10 7 gc/L; plant 2) of wastewater samples. The results of the study demonstrate the frequent detection of two primary respiratory viruses in wastewater and offer the possibility to track the epidemiology of influenza by wastewater-based monitoring.

Viral evolution refers to the genetic changes that a virus accumulates during its lifetime which can arise from adaptations in response to environmental changes or the immune response of the host. Influenza A virus is one of the most rapidly evolving microorganisms. Its genetic instability may lead to large changes in its biological properties, including changes in virulence, adaptation to new hosts, and even the emergence of infectious diseases with a previously unknown clinical course. Genetic variability makes it difficult to implement effective prophylactic programs, such as vaccinations, and may be responsible for resistance to antiviral drugs. The aim of the review was to describe the consequences of the variability of influenza viruses, mutations, and recombination, which allow viruses to overcome species barriers, causing epidemics and pandemics. Another consequence of influenza virus evolution is the risk of the resistance to antiviral drugs. Thus far, one class of drugs, M2 protein inhibitors, has been excluded from use because of mutations in strains isolated in many regions of the world from humans and animals. Therefore, the effectiveness of anti-influenza drugs should be continuously monitored in reference centers representing particular regions of the world as a part of epidemiological surveillance., Competing Interests: The authors declare no conflicts of interest.

When two influenza viruses co-infect the same cell, they can exchange genome segments in a process known as reassortment. Reassortment is an important source of genetic diversity and is known to have been involved in the emergence of most pandemic influenza strains. However, because of the difficulty in identifying reassortment events from viral sequence data, little is known about their role in the evolution of the seasonal influenza viruses. Here we introduce TreeKnit, a method that infers ancestral reassortment graphs (ARG) from two segment trees. It is based on topological differences between trees, and proceeds in a greedy fashion by finding regions that are compatible in the two trees. Using simulated genealogies with reassortments, we show that TreeKnit performs well in a wide range of settings and that it is as accurate as a more principled bayesian method, while being orders of magnitude faster. Finally, we show that it is possible to use the inferred ARG to better resolve segment trees and to construct more informative visualizations of reassortments., Competing Interests: The authors have declared that no competing interests exist.

Current Situation: The global influenza surveillance and response system (GISRS), coordinated by the World Health Organization (WHO), is a global framework for surveillance of influenza and other respiratory viruses, data collection, laboratory capacity building, genomic data submission and archival, standardization, and calibration of reagents and vaccine strains, production of seasonal influenza vaccines and creating a facilitatory regulatory environment for the same. GAPS: WHO-designated national influenza centers (NICs) are entrusted with establishing surveillance in their respective countries. National and subnational surveillance remains weak in most parts of the world because of varying capacities of the NICs, lack of funds, poor human and veterinary surveillance mechanisms, lack of intersectoral coordination, and varying commitments of the local government., Way Forward: As influenza viruses have a wide variety of nonhuman hosts, it is critical to strengthen surveillance at local levels for timely detection of untypable or novel strains with potential to cause epidemics or pandemics. In this article, we have proposed possible strategies to strengthen and expand local capacities for respiratory virus surveillance through the designated NICs of the WHO., Competing Interests: Conflict of interest The authors have no competing interest to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

It is well known that the difficulty of siRNA therapeutic application is the lack of safe and effective delivery vector. Virosome is a nano vesicle composed of lipid membrane and membrane protein. It retains fusion protein without virus genetic material, and therefore has the reduced immunogenicity compared with viral vector. Virosomes have the potential to deliver protein and nucleic acid drugs, but the traditional preparation method of virosomes is quite limited. In this study, we firstly proposed to synthesize influenza virus hemagglutinin HA2 virosomes by cell-free protein synthesis. In this study, liposomes provided the hydrophobic lipid bilayer environment for the formation of HA2 protein multimer, which inhibited the aggregation of hydrophobic HA2 and improved HA2 protein expression. Chitosan as a rigid core adsorbed siRNA and improved the encapsulation efficiency of siRNA. In conclusion, the cell-free protein synthesis was used to prepare HA2 virosomes, which paves the way for constructing a novel nano vector with high delivery efficiency and biosafety for the delivery of siRNA., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Respiratory illness caused by influenza virus is a serious public health problem worldwide. As the symptoms of influenza virus infection are similar to those of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is essential to distinguish these two viruses. Therefore, to properly respond to a pathogen, a detection method that is capable of rapid and accurate diagnosis in a hospital or at home is required. To satisfy this need, we applied loop-mediated isothermal amplification (LAMP) and an isothermal nucleic acid amplification technique, along with a system to analyze the results without specialized equipment, a lateral flow assay (LFA). Using the platform developed in this study, all processes, from sample preparation to detection, can be performed without special equipment. Unlike existing PCR methods, the nucleic acid amplification can be performed in the field because hot packs do not require electricity. Thus, the designed platform can provide rapid results without the need to transport the samples to a laboratory or hospital. These advantages are not limited to operations in developing countries with poor access to medical systems. In conclusion, the developed technology is a promising tool for infectious disease management that allows for rapid identification of infectious diseases and appropriate treatment of patients., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: During the coronavirus disease 2019 (COVID-19) pandemic, seasonal influenza activity declined globally and remained below previous seasonal levels, but intensified in China since 2021. Preventive measures to COVID-19 accompanied by different epidemic characteristics of influenza in different regions of the world. To better respond to influenza outbreaks under the COVID-19 pandemic, we analyzed the epidemiology, antigenic and genetic characteristics, and antiviral susceptibility of influenza viruses in the mainland of China during 2020-2021., Methods: Respiratory specimens from influenza like illness cases were collected by sentinel hospitals and sent to network laboratories in Chinese National Influenza Surveillance Network. Antigenic mutation analysis of influenza virus isolates was performed by hemagglutination inhibition assay. Next-generation sequencing was used for genetic analyses. We also conducted molecular characterization and phylogenetic analysis of circulating influenza viruses. Viruses were tested for resistance to antiviral medications using phenotypic and/or sequence-based methods., Results: In the mainland of China, influenza activity recovered in 2021 compared with that in 2020 and intensified during the traditional influenza winter season, but it did not exceed the peak in previous years. Almost all viruses isolated during the study period were of the B/Victoria lineage and were characterized by genetic diversity, with the subgroup 1A.3a.2 viruses currently predominated. 37.8% viruses tested were antigenically similar to reference viruses representing the components of the vaccine for the 2020-2021 and 2021-2022 Northern Hemisphere influenza seasons. In addition, China has a unique subgroup of 1A.3a.1 viruses. All viruses tested were sensitive to neuraminidase inhibitors and endonuclease inhibitors, except two B/Victoria lineage viruses identified to have reduced sensitivity to neuraminidase inhibitors., Conclusions: Influenza activity increased in the mainland of China in 2021, and caused flu season in the winter of 2021-2022. Although the diversity of influenza (sub)type decreases, B/Victoria lineage viruses show increased genetic and antigenic diversity. The world needs to be fully prepared for the co-epidemic of influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus globally., (© 2022. The Author(s).)

The COVID-19 pandemic required increased testing capacity, enabling rapid case identification and effective contract tracing to reduce transmission of disease. The BioFire FilmArray is a fully automated nucleic acid amplification test system providing specificity and sensitivity associated with gold standard molecular methods. The FilmArray Respiratory Panel 2.1 targets 22 viral and bacterial pathogens, including SARS-CoV-2 and influenza virus. While each panel provides a robust output of information regarding pathogen detection, the specimen throughput is low. This study evaluates the FilmArray Respiratory Panel 2.1 using 33 pools of contrived nasal samples and 22 pools of clinical nasopharyngeal specimens to determine the feasibility of increasing testing capacity, while maintaining detection of both SARS-CoV-2 and influenza virus. We observed 100% detection and 90% positive agreement for SARS-CoV-2 and 98% detection and 95% positive agreement for influenza viruses with pools of contrived or clinical specimens, respectively. While discordant results were mainly attributed to loss in sensitivity, the sensitivity of the pooling assay was well within accepted limits of detection for a nucleic acid amplification test. Overall, this study provides evidence supporting the use of pooling patient specimens, one in four with the FilmArray Respiratory Panel 2.1 for the detection of SARS-CoV-2 and influenza virus., (© 2022. The Author(s).)

Background: In the context of COVID-19 pandemic in Catalonia (Spain), the present study analyses respiratory samples collected by the primary care network using Acute Respiratory Infections Sentinel Surveillance System (PIDIRAC) during the 2019-2020 season to complement the pandemic surveillance system in place to detect SARS-CoV-2. The aim of the study is to describe whether SARS-CoV-2 was circulating before the first confirmed case was detected in Catalonia, on February 25th, 2020., Methods: The study sample was made up of all samples collected by the PIDIRAC primary care network as part of the Influenza and Acute Respiratory Infections (ARI) surveillance system activities. The study on respiratory virus included coronavirus using multiple RT-PCR assays. All positive samples for human coronavirus were subsequently typed for HKU1, OC43, NL63, 229E. Every respiratory sample was frozen at-80°C and retrospectively studied for SARS-CoV-2 detection. A descriptive study was performed, analysing significant differences among variables related to SARS-CoV- 2 cases comparing with rest of coronaviruses cases through a bivariate study with Chi-squared test and statistical significance at 95%., Results: Between October 2019 and April 2020, 878 respiratory samples from patients with acute respiratory infection or influenza syndrome obtained by PIDIRAC were analysed. 51.9% tested positive for influenza virus, 48.1% for other respiratory viruses. SARS-CoV-2 was present in 6 samples. The first positive SARS-CoV-2 case had symptom onset on 2 March 2020. These 6 cases were 3 men and 3 women, aged between 25 and 50 years old. 67% had risk factors, none had previous travel history nor presented viral coinfection. All of them recovered favourably., Conclusion: Sentinel Surveillance PIDIRAC enhances global epidemiological surveillance by allowing confirmation of viral circulation and describes the epidemiology of generalized community respiratory viruses' transmission in Catalonia. The system can provide an alert signal when identification of a virus is not achieved in order to take adequate preparedness measures., Competing Interests: The author declares that neither they nor any of the co-authors have a conflict of interest regarding the publication of this manuscript.

Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, limiting their efficiency and utility in high-throughput applications. More streamlined in silico tools allowing researchers to properly analyze and visualize influenza viral protein sequences with accurate nomenclature are necessary to improve antigen design and productivity. To address this need, we developed Librator, a system for analyzing and designing custom protein sequences of influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Within Librator's graphical interface, users can easily interrogate viral sequences and phylogenies, visualize antigen structures and conservation, mutate target residues and design custom antigens. Librator also provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible across related subtypes, thereby promoting reagent savings. Finally, the program facilitates single-cell immune profiling, epitope mapping of monoclonal antibodies and mosaic protein design. Using Librator-based antigen construction, we demonstrate that antigenicity can be readily transferred between HA molecules of H3, but not H1, lineage viruses. Altogether, Librator is a valuable tool for analyzing influenza virus HA and NA proteins and provides an efficient resource for optimizing recombinant influenza antigen synthesis., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands)., Competing Interests: The authors have declared that no competing interests exist.