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Prim-O-glucosylcimifugin (POG) is a chromone derived primarily from Saposhnikovia divaricata (Turcz) Schischk and Cimicifuga simplex. Previous research has shown that POG possesses antibacterial, anticancer, anti-inflammatory, antioxidant, anticonvulsant, antipyretic, and analgesic properties. However, the specific impact of POG on influenza-virus-induced pneumonia is not well understood. In this study, we investigated the protective effects and underlying mechanisms of POG in pneumonia caused by influenza A virus (IAV). In vitro, POG was found to have a protective effect against infections caused by the respiratory viruses respiratory syncytial virus (RSV), human coronavirus OC43, and influenza A virus. POG inhibited A/FM/1/1947(H1N1) infection with an EC50 ranging from 3.01 to 10.43 in vitro. Intraperitoneal infection of mice with POG at a dose of 5 or 10 mg/kg resulted in a reduction in IAV-induced pneumonia, as evidenced by decreased pulmonary edema, improved lung histopathology, and reduced inflammatory cell accumulation. At the higher dose (10 mg/kg), POG treatment significantly increased survival rates, decreased viral titres in the lungs, improved lung histology, and reduced lung inflammation in IAV-infected mice. POG also effectively alleviated pulmonary fibrosis by reducing the levels of fibrotic markers (hydroxyproline [Hyp] and transforming growth factor β1 [TGF-β1]) and suppressing the expression of alpha smooth muscle actin (α-SMA), p focal adhesion kinase (p-FAK), and TGF-β1 in lung tissues. In addition, POG inhibited the expression of the RELA proto-oncogene (RELA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), and mitogen- and stress-activated protein kinase 2 (MSK2) in lung tissues. These results indicate that POG may have a protective effect against IAV-induced pneumonia by downregulating the TGF-β1/PI3KCD/MSK2/RELA signalling pathway in the lungs. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies are an effective method of treating acute infectious diseases. However, knowing which of the produced antibodies in the vast number of human antibodies can cure the disease requires a long time and advanced technology. The previously introduced iBRAB method relies on studied antibodies to design a broad-spectrum antibody capable of neutralizing antigens of many different Influenza A viral strains. To evaluate the antigen-binding fragment as an applicable drug, the therapeutic antibody profiles providing guidelines collected from clinically staged therapeutic antibodies were used to access different measurements. Although the evaluated values were within an accepted range, the modification in the amino acid sequence is required for better properties. Thus, using the steered molecular dynamics (SMD) simulation to determine the binding capacity of amino acids in the functional region, the profile of interacted amino acids of Fab with the antigen was established for modified reference. As a result, the model was modified with amino acids elimination at positions 96–97 in the heavy chain and 26–27, 91, 96–97, and 102–103 in the light chain, which has better Therapeutic Antibody Profiler evaluations than the original designation. Thus again, SMD simulation is a promising computational approach for post-modification in rational drug design. [ABSTRACT FROM AUTHOR]

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket. Modulation of calcium binding in the neuraminidase low-affinity calcium-binding pocket suggests a novel role of calcium during cross-species transmission of Influenza A viruses. [ABSTRACT FROM AUTHOR]

Prior research has indicated that the gut-lung-axis can be influenced by the intestinal microbiota, thereby impacting lung immunity. Rifaximin is a broad-spectrum antibacterial drug that can maintain the homeostasis of intestinal microflora. In this study, we established an influenza A virus (IAV)-infected mice model with or without rifaximin supplementation to investigate whether rifaximin could ameliorate lung injury induced by IAV and explore the molecular mechanism involved. Our results showed that IAV caused significant weight loss and disrupted the structure of the lung and intestine. The analysis results of 16S rRNA and metabolomics indicated a notable reduction in the levels of probiotics Lachnoclostridium, Ruminococcaceae_UCG-013, and tryptophan metabolites in the fecal samples of mice infected with IAV. In contrast, supplementation with 50 mg/kg rifaximin reversed these changes, including promoting the repair of the lung barrier and increasing the abundance of Muribaculum, Papillibacter and tryptophan-related metabolites content in the feces. Additionally, rifaximin treatment increased ILC3 cell numbers, IL-22 level, and the expression of RORγ and STAT-3 protein in the lung. Furthermore, our findings demonstrated that the administration of rifaximin can mitigate damage to the intestinal barrier while enhancing the expression of AHR, IDO-1, and tight junction proteins in the small intestine. Overall, our results provided that rifaximin alleviated the imbalance in gut microbiota homeostasis induced by IAV infection and promoted the production of tryptophan-related metabolites. Tryptophan functions as a signal to facilitate the activation and movement of ILC3 cells from the intestine to the lung through the AHR/STAT3/IL-22 pathway, thereby aiding in the restoration of the barrier. Key points: • Rifaximin ameliorated IAV infection-caused lung barrier injury and induced ILC3 cell activation. • Rifaximin alleviated IAV-induced gut dysbiosis and recovered tryptophan metabolism. • Tryptophan mediates rifaximin-induced ILC3 cell activation via the AHR/STAT3/IL-22 pathway. [ABSTRACT FROM AUTHOR]

In March 2023, our pediatric intensive care unit (PICU) retrospectively examined six cases of pediatric necrotizing tracheobronchitis (NTB), focusing on co-infections with influenza A virus (IAV) and Staphylococcus aureus (S. aureus). This study aimed to elucidate NTB's clinical characteristics, diagnostics, and therapeutic approaches. Diagnostics included symptom assessment, microbiological testing that confirmed all patients were positive for IAV H1N1 with a predominant S. aureus co-infection, and bronchoscopy. The patients predominantly exhibited fever, cough, and dyspnea. Laboratory analysis revealed decreased lymphocyte counts and elevated infection markers like C-reactive protein and procalcitonin. Chest computed tomography (CT) scans detected tracheobronchial obstructions in half of the cases, while bronchoscopy showed severe mucosal congestion, edema, necrosis, and purulent-hemorrhagic exudates. Treatments encompassed comprehensive strategies like oxygen therapy, intubation, bronchoscopic interventions, thoracentesis, oseltamivir, and a regimen of antibiotics. Our findings suggested potential correlations between clinical markers, notably lymphocyte count and procalcitonin, and clinical interventions such as the number of rescues and intensive care unit (ICU) duration. This research highlights the importance of early detection and the role of bronchoscopy and specific markers in assessing NTB, advocating for continued research in larger cohorts to better understand its clinical trajectory and refine treatment approaches for this challenging pediatric disease. [ABSTRACT FROM AUTHOR]

Influenza A virus infection, commonly known as the flu, has persisted in the community for centuries. Although we have yearly vaccinations to prevent seasonal flu, there remains a dire need for antiviral drugs to treat active infections. The constantly evolving genome of the influenza A virus limits the number of effective antiviral therapeutic options. Over time, antiviral drugs become inefficient due to the development of resistance, as seen with adamantanes, which are now largely ineffective against most circulating strains of the virus. Neuraminidase inhibitors have long been the drug of choice, but due to selection pressure, strains are becoming resistant to this class of drugs. Baloxavir marboxil, a drug with a novel mode of action, can be used against strains resistant to other classes of drugs but is still not available in many countries. Deep research into nanoparticles has shown they are effective as antiviral drugs, opening a new avenue of research to use them as antiviral agents with novel modes of action. As this deadly virus, which has killed millions of people in the past, continues to develop resistance, there is an urgent need for new therapeutic agents with novel modes of action to halt active infections in patients. This review article covers the available therapeutic antiviral drug options with different modes of action, their effectiveness, and resistance to various strains of influenza A virus. [ABSTRACT FROM AUTHOR]

Studies on the immune-regulatory roles played by the commensal microbes residing in the nasal mucosa consider the contribution of antiviral immune responses. Here, we sought to identify the nasal microbiome, Staphylococcus epidermidis-regulated antiviral immune responses and the alteration of polyamine metabolites in nasal epithelium. We found that polyamines were required for the life cycle of influenza A virus (IAV) and depletion of polyamines disturbed IAV replication in normal human nasal epithelial (NHNE) cells. Inoculation of S. epidermidis also suppressed IAV infection and the concentration of polyamines including putrescine, spermidine, and spermine was completely attenuated in S. epidermidis-inoculated NHNE cells. S. epidermidis activated the enzyme involved in the production of ornithine from arginine and downregulated the activity of the enzyme involved in the production of putrescine from ornithine in nasal epithelium. S. epidermidis also induced the activation of enzymes that promote the extracellular export of spermine and spermidine in NHNE cells. Our findings demonstrate that S. epidermidis is shown to be able of creating an intracellular environment lacking polyamines in the nasal epithelium and promote the balance of cellular polyamines in favor of the host to restrict influenza virus replication. Staphylococcus epidermidis, a nasal commensal in healthy mucus, can create an intracellular epithelium environment lacking polyamines and promote balance of cellular polyamines in favor of the host to restrict influenza virus replication. [ABSTRACT FROM AUTHOR]

In 2004, the equine-origin H3N8 canine influenza virus (CIV) first caused an outbreak with lethal cases in racing greyhounds in Florida, USA, and then spread to domestic dogs nationwide. Although transmission of this canine virus to humans has not been reported, it is important to evaluate its zoonotic potential because of the high contact opportunities between companion dogs and humans. To gain insight into the interspecies transmissibility of H3N8 CIV, we tested its adaptability to human respiratory A549 cells through successive passages. We found that CIV acquired high growth properties in these cells mainly through mutations in surface glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). Our reverse genetics approach revealed that HA2-K82E, HA2-R163K, and NA-S18L mutations were responsible for the increased growth of CIV in human cells. Molecular analyses revealed that both HA2 mutations altered the optimum pH for HA membrane fusion activity and that the NA mutation changed the HA-NA functional balance. These findings suggest that H3N8 CIV could evolve into a human pathogen with pandemic potential through a small number of mutations, thereby posing a threat to public health in the future. [ABSTRACT FROM AUTHOR]

Influenza virus infection remains a major global health problem and requires a universal vaccine with broad protection against different subtypes as well as a rapid-response vaccine to provide immediate protection in the event of an epidemic outbreak. Here, we show that intranasal administration of probiotic Escherichia coli Nissle 1917 activates innate immunity in the respiratory tract and provides immediate protection against influenza virus infection within 1 day. Based on this vehicle, a recombinant strain is engineered to express and secret five tandem repeats of the extracellular domain of matrix protein 2 from different influenza virus subtypes. Intranasal vaccination with this strain induces durable humoral and mucosal responses in the respiratory tract, and provides broad protection against the lethal challenge of divergent influenza viruses in female BALB/c mice. Our findings highlight a promising delivery platform for developing mucosal vaccines that provide immediate and sustained protection against respiratory pathogens. Influenza virus infection is a global health threat and vaccines are required that show broad protection against a range of viral subtypes. Here the authors present a universal influenza vaccine based on Escherichia coli Nissle 1917 that activates innate and adaptive humoral and mucosal immunity, providing both immediate and long-term protection against influenza A virus infection in a murine model. [ABSTRACT FROM AUTHOR]

In vivo assessments of influenza A virus (IAV) pathogenicity and transmissibility in ferrets represent a crucial component of many pandemic risk assessment rubrics, but few systematic efforts to identify which data from in vivo experimentation are most useful for predicting pathogenesis and transmission outcomes have been conducted. To this aim, we aggregated viral and molecular data from 125 contemporary IAV (H1, H2, H3, H5, H7, and H9 subtypes) evaluated in ferrets under a consistent protocol. Three overarching predictive classification outcomes (lethality, morbidity, transmissibility) were constructed using machine learning (ML) techniques, employing datasets emphasizing virological and clinical parameters from inoculated ferrets, limited to viral sequence-based information, or combining both data types. Among 11 different ML algorithms tested and assessed, gradient boosting machines and random forest algorithms yielded the highest performance, with models for lethality and transmission consistently better performing than models predicting morbidity. Comparisons of feature selection among models was performed, and highest performing models were validated with results from external risk assessment studies. Our findings show that ML algorithms can be used to summarize complex in vivo experimental work into succinct summaries that inform and enhance risk assessment criteria for pandemic preparedness that take in vivo data into account. Use of machine learning to summarize complex in vivo data and provide predictive utility for Influenza A virus ferret lethality, morbidity, and transmission models. [ABSTRACT FROM AUTHOR]

Objective: To investigate the clinical characteristics and risk factors for pneumonia in children co-infected with influenza A virus (IAV) and Mycoplasma pneumoniae (MP). Methods: Children who were diagnosed with IAV and MP infection between January and December, 2023 were enrolled and divided into a non-pneumonia group and a pneumonia group. Univariate analysis and logistic regression analysis were used to evaluate each index, and the risk factors for pneumonia caused by coinfection in the two groups were explored. Results: A total of 209 patients were enrolled, of which 107 and 102 patients were in the pneumonia and non-pneumonia groups, respectively. The patients in the pneumonia group were older and had a longer duration of fever (P < 0.05). Univariate analysis revealed that the median age, duration of fever, and CD3+, CD4+, CD8+ and IL-10 levels were significantly correlated with pneumonia (P < 0.05). Multivariate logistic regression analysis revealed that the median age, duration of fever, and CD4+, CD8+ and IL-10 levels were independent risk factors for pneumonia. Area under the curve of the five combined indicators in the ROC (receiver operator characteristic) analysis was 0.883, was higher than single factor. Conclusion: Children with IAV and MP infection whose age older than 6.08 years, had a fever longer than 4 days, had a CD4+ count < 22.12%, had a CD8+ count < 35.21%, had an IL-10 concentration > 22.08 ng/ml were more likely to develop pneumonia. [ABSTRACT FROM AUTHOR]

By modifying ring A in the dammarane triterpenoid dipterocarpol, derivatives with isoxazole and 2-cyano-1-ene fragments were synthesized, which showed activity against the influenza A virus (H1N1) with selectivity indices of 19 and 23. [ABSTRACT FROM AUTHOR]

Influenza A virus has a wide natural areal among birds, mammals, and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian–swine origin but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of adaptation of avian influenza viruses to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures. [ABSTRACT FROM AUTHOR]

Pyridine-containing amides of rimantadine (1-(1-adamantyl)ethylamine) were synthesized by the amidation of rimantadine hydrochloride by the corresponding acids in the NEt3—N,N′-dicyclohexylcarbodiimide (DCC)—4-(N,N-dimethylamino)pyridine (DMAP) system as the analogues of previously described antiviral compounds 1-adamantylmethyl-5-aminoisoxazole-3-carboxylate and N-{[5-(thien-2-yl)isoxazol-3-yl]methyl}adamantane-1-amine. The spectrum of antiviral activity against the rimantadine-resistant A/PR/8/34 strain of influenza A virus, tick-borne encephalitis virus (TBEV) strain Absettarov (European subtype), yellow fever virus (YFV) vaccine strain 17D, and West Nile virus (WNV) strain Strix nebulosa-12 was studied. The structure—activity relationships were discussed. Synthesized compounds efficiently inhibited the reproduction of TBEV and WNV. N-[1-(Adamantan-1-yl)ethyl]picolinamide efficiently inhibited the reproduction of enveloped viruses with both type I (influenza) and type II (flaviviruses) fusion proteins. The antiviral activity against the rimantadine-resistant influenza virus A/PR/8/34 and a low cytotoxicity (high selectivity index) were revealed for N-[1-(adamantan-1-yl)ethyl]-isonicotinamide. [ABSTRACT FROM AUTHOR]

New homodimers of 1,2,3-triazolyl nucleoside analogs based on 6-methyluracil, which are linked by the polymethylene linker at the N(1) or N(3) atoms, were synthesized. Screening of in vitro antiviral activity against influenza A virus (H1N1) and Coxsackievirus B3 revealed the lead compound that in vitro inhibited the replication of Coxsackievirus B3 with a half-maximal inhibitory concentration (IC50) and a half-maximal cytotoxic concentration (CC50) of 30.1 and >374 µmol L−1, respectively. A dependence of the antiviral activity on the length of the polymethylene linker connecting the 6-methyluracil and 1,2,3-triazolyl-β-d-ribofuranosyl fragments was found. [ABSTRACT FROM AUTHOR]

Objectives: The concurrent impact of COVID-19 and influenza on disease burden is a topic of great concern. This discussion delves into the epidemiological characteristics of seasonal influenza activity in Shanghai within the context of the SARS-CoV-2 epidemic. Methods: From 2017 to 2023, a total of 11,081 patients having influenza-like illness (ILI) were included in this study for influenza virus detection. Reverse transcription polymerase chain reaction (RT-PCR) assays were conducted according to standardised protocols to identify the types and subtypes of influenza viruses. The positivity rate of the influenza virus among the sampled ILI cases served as a surrogate measure for estimating various influenza seasonal characteristics, such as periodicity, duration, peak occurrences, and the prevalent subtypes or lineages. Epidemiological aspects across different years and age groups were subjected to comprehensive analysis. For categorical variables, the Chi-square test or Fisher's exact test was employed, as deemed appropriate. Results: A total of 1553 (14.0%) tested positive for influenza virus pathogens. The highest positivity rate for influenza was observed in adults aged 25–59 years (18.8%), while the lowest rate was recorded in children under 5 years (3.8%). The influenza circulation patterns in Shanghai were characterised: (1) 2 years exhibited semiannual periodicity (2017–2018, 2022–2023); (2) 3 years displayed annual periodicity (2018–2019, 2019–2020, and 2021–2022); and (3) during 2020–2021, epidemic periodicities of seasonal influenza viruses disappeared. In terms of influenza subtypes, four subtypes were identified during 2017–2018. In 2018–2019 and 2019–2020, A/H3N2, A/H1N1, and B/Victoria were circulating. Notably, one case of B/Victoria was detected in 2020–2021. The epidemic period of 2021–2022 was attributed to B/Victoria, and during 2022–2023, the influenza A virus was the dominant circulating strain. Conclusions: The seasonal epidemic period and the predominant subtype/lineage of influenza viruses around the SARS-CoV-2 epidemic period in Shanghai city are complex. This underscores the necessity for vigilant influenza control strategies amidst the backdrop of other respiratory virus pandemics. [ABSTRACT FROM AUTHOR]

Current Influenza virus vaccines primarily induce antibody responses against variable epitopes in hemagglutinin (HA), necessitating frequent updates. However, antibodies against neuraminidase (NA) can also confer protection against influenza, making NA an attractive target for the development of novel vaccines. In this study, we aimed to enhance the immunogenicity of recombinant NA antigens by presenting them multivalently on a nanoparticle carrier. Soluble tetrameric NA antigens of the N1 and N2 subtypes, confirmed to be correctly folded by cryo-electron microscopy structural analysis, were conjugated to Mi3 self-assembling protein nanoparticles using the SpyTag-SpyCatcher system. Immunization of mice with NA-Mi3 nanoparticles induced higher titers of NA-binding and -inhibiting antibodies and improved protection against a lethal challenge compared to unconjugated NA. Additionally, we explored the co-presentation of N1 and N2 antigens on the same Mi3 particles to create a mosaic vaccine candidate. These mosaic nanoparticles elicited antibody titers that were similar or superior to the homotypic nanoparticles and effectively protected against H1N1 and H3N2 challenge viruses. The NA-Mi3 nanoparticles represent a promising vaccine candidate that could complement HA-directed approaches for enhanced potency and broadened protection against influenza A virus. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) causes significant morbidity and mortality worldwide due to seasonal epidemics and periodic pandemics. The antigenic drift/shift of IAV continually gives rise to new strains and subtypes, aiding IAV in circumventing previously established immunity. As a result, there has been substantial interest in developing a broadly protective IAV vaccine that induces, durable immunity against multiple IAVs. Previously, a polyanhydride nanoparticle-based vaccine or nanovaccine (IAV-nanovax) encapsulating H1N1 IAV antigens was reported, which induced pulmonary B and T cell immunity and resulted in cross-strain protection against IAV. A key feature of IAV-nanovax is its ability to easily incorporate diverse proteins/payloads, potentially increasing its ability to provide broad protection against IAV and/or other pathogens. Due to human susceptibility to both H1N1 and H3N2 IAV, several H3N2 nanovaccines were formulated herein with multiple IAV antigens to examine the "plug-and-play" nature of the polyanhydride nanovaccine platform and determine their ability to induce humoral and cellular immunity and broad-based protection similar to IAV-nanovax. The H3N2-based IAV nanovaccine formulations induced systemic and mucosal B cell responses which were associated with antigen-specific antibodies. Additionally, systemic and lung-tissue resident CD4 and CD8 T cell responses were enhanced post-vaccination. These immune responses corresponded with protection against both homologous and heterosubtypic IAV infection. Overall, these results demonstrate the plug-and-play nature of the polyanhydride nanovaccine platform and its ability to generate immunity and protection against IAV utilizing diverse antigenic payloads. [ABSTRACT FROM AUTHOR]

Data from influenza A virus (IAV) infected ferrets provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard model can recapitulate many clinical signs of infection present in IAV-infected humans, support virus replication of human, avian, swine, and other zoonotic strains without prior adaptation, and permit evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible. Here, we describe a dataset of 728 ferrets inoculated with 126 unique IAV, conducted by a single research group under a uniform experimental protocol. This collection of morbidity, mortality, and viral titer data represents the largest publicly available dataset to date of in vivo-generated IAV infection outcomes on a per-ferret level. [ABSTRACT FROM AUTHOR]

With the lifting of COVID-19 non-pharmaceutical interventions, the resurgence of common viral respiratory infections was recorded in several countries worldwide. It facilitates viral co-infection, further burdens the already over-stretched healthcare systems. Racing to find co-infection-associated efficacy therapeutic agents need to be rapidly established. However, it has encountered numerous challenges that necessitate careful investigation. Here, we introduce a potential recombinant minibody-associated treatment, 3D8 single chain variable fragment (scFv), which has been developed as a broad-spectrum antiviral drug that acts via its nucleic acid catalytic and cell penetration abilities. In this research, we demonstrated that 3D8 scFv exerted antiviral activity simultaneously against both influenza A viruses (IAVs) and coronaviruses in three established co-infection models comprising two types of coronaviruses [beta coronavirus—human coronavirus OC43 (hCoV-OC43) and alpha coronavirus—porcine epidemic diarrhea virus (PEDV)] in Vero E6 cells, two IAVs [A/Puerto Rico/8/1934 H1N1 (H1N1/PR8) and A/X-31 (H3N2/X-31)] in MDCK cells, and a combination of coronavirus and IAV (hCoV-OC43 and adapted-H1N1) in Vero E6 cells by a statistically significant reduction in viral gene expression, proteins level, and approximately around 85%, 65%, and 80% of the progeny of 'hCoV-OC43–PEDV', 'H1N1/PR8–H3N2/X-31', and 'hCoV-OC43–adapted-H1N1', respectively, were decimated in the presence of 3D8 scFv. Taken together, we propose that 3D8 scFv is a promising broad-spectrum drug for treatment against RNA viruses in co-infection. [ABSTRACT FROM AUTHOR]

Prototypic receptors for human influenza viruses are N-glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin's (HAs) are examined by chemoenzymatic synthesis of N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD-1H by 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding. Binding modes of antigenically drifted hemagglutinins of human influenza A viruses have been determined by NMR using synthetic N-glycans having 13C-labeled monosaccharides to pinpoint which monosaccharides of extended LacNAc chains engage with the HAs. [ABSTRACT FROM AUTHOR]

In this paper, the possibility of detecting viruses, specifically influenza A virus, based on changes in the spectra of total reflection from macroporous silicon (macro-pSi) films, is demonstrated for the first time. Macro-pSi films with a pore diameter of about 100 nm were produced by electrochemical etching of crystalline silicon substrates. The porosity of the macro-pSi, calculated using the Bruggeman effective medium model, was 75%. Electron microscopy showed that such highly porous films adsorb of 50–100 nm in size viruses on their surface and inside the pores, but the efficiency of adsorption significantly increases when the surface of the nanostructures is functionalized with monoclonal antibodies, providing specific binding of viruses. The reflection spectra of macro-pSi films demonstrate a series of interference fringes, the intensity of which dramatically changes upon virus adsorption. The results obtained demonstrate the possibility of a simple and effective optical method for virus diagnostics using Fabry–Pérot interference in macro‑pSi films. [ABSTRACT FROM AUTHOR]

Characterizing the mutational diversity of viruses can shed light on both intra-host and between-host evolutionary processes. In this study, we analyzed the transcriptome profiles of five different strains of Influenza A virus H1N1 isolated from humans, ducks, and pigs. We detected positive selection that led to the evolution and fixation of virus variants at the intra-host level. Among the strains studied, A/mallard/Balkhash/6304/2014 was the most affected by intra-host dynamics, with 143 substitutions, while A/Puerto Rico/8/34 (PR8) had the least, with only one substitution. A/California/04/09 had the highest frequency of distribution in the HA epitope sites. We also detected amino acid substitutions in immunodominant antigenic regions of A/WSN/1933, mallard, and swine influenza strains. We detected a total of 19 shared substitutions across both the inter-host and intra-host levels. The sequences of the different strains showed different patterns of gene expression. Phylogenetic trees constructed based on the HA and NA segments of the 26 GenBank isolates showed incongruences with other phylogenetic trees based on other IAV segments. We also identified amino acid changes in critical regions of the studied isolates, which could provide valuable information on the contingency of novel recombinants with different morphogenesis, infectivity, and behavior against antivirals. Our results show that influenza A viruses (IAVs) could potentially adapt more efficiently within their host organisms, mostly in wild birds. This underlines the importance of certain population characteristics, such as the elimination of genetic bottlenecks, as a means to expand their ability to infect new hosts. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR–HA interaction in the fusion of viral and late endosomal membranes during IAV replication. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the long-term effects of non-pharmacological interventions (NPIs) measures on the epidemiological characteristics of common respiratory viruses in preschool children in Henan, China. Methods: This was a retrospective observational study containing data from 17 prefecture-level cities in Henan, China. We analyzed and compared laboratory results and clinical data of preschool children presenting to outpatient clinics for acute respiratory infections (ARTI) after COVID-19 (January 2020–October 2022) and before COVID-19 (December 2017–December 2019). Each year was divided into quarters. The ratio of the odds ratios (ORs) of testing positive for eight respiratory viruses in each year after the pandemic to the prepandemic period was estimated applying a generalized linear model (GLM), using the mean of the positive detection rates in 2018–2019 as a reference. Results: A total of 11,400 children were enrolled from December 2017 to October 2022. The number of positive detections for all respiratory viruses decreased in 2020–2022 compared to the average of 2018–2019. Human respiratory syncytial virus (hRSV), human rhinovirus (hRV), and influenza virus (IFV) accounted for a larger proportion of all detected viruses before COVID-19 pandemic, whereas hRV, human bocavirus (hBoV), and human adenovirus (hAdV) accounted for a significantly larger proportion after COVID-19 pandemic. The positive detection rates of enveloped viruses [IFV, human parainfluenza virus (hPIV), hRSV, human metapneumovirus (hMPV), and human coronavirus (hCoV)] decreased sharply and the seasonal activity of these viruses was weakened, while the positive detection rates of non-enveloped viruses (hRV, hBoV, and hAdV) increased, especially hRV. The conditions described above tended to occur more frequently in boys and children older than 1 year, and they were also more sensitive to the NPIs. Conclusions: NPIs transformed the epidemiological profile of common respiratory viruses among preschool children during the COVID-19 pandemic. To improve the overall public health response to all respiratory viruses, interventions targeting non-enveloped viruses need to be strengthened to mitigate their continued transmission. [ABSTRACT FROM AUTHOR]

At the end of 2020, an outbreak of HPAI H5N8 was registered in captive African houbara bustards (Chlamydotis undulata) in the United Arab Emirates. In order to better understand the pathobiology of this viral infection in bustards, a comprehensive pathological characterization was performed. A total of six birds were selected for necropsy, histopathology, immunohistochemistry, RNAscope in situ hybridization and RT-qPCR and nanopore sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue blocks. Gross lesions included mottled and/or hemorrhagic pancreas, spleen and liver and fibrinous deposits on air sacs and intestine. Necrotizing pancreatitis, splenitis and concurrent vasculitis, hepatitis and fibrino-heterophilic peritonitis were identified, microscopically. Viral antigens (nucleoprotein) and RNAs (matrix gene) were both detected within necro-inflammatory foci, parenchymal cells, stromal cells and endothelial cells of affected organs, including the myenteric plexus. Molecular analysis of FFPE blocks successfully detected HPAI H5N8, further confirming its involvement in the lesions observed. In conclusion, HPAI H5N8 in African houbara bustards results in hyperacute/acute forms exhibiting marked pantropism, endotheliotropism and neurotropism. In addition, our findings support the use of FFPE tissues for molecular studies of poorly characterized pathogens in exotic and endangered species, when availability of samples is limited. [ABSTRACT FROM AUTHOR]

Defective interfering particles (DIPs) of influenza A virus (IAV) are suggested for use as broad-spectrum antivirals. We discovered a new type of IAV DIP named "OP7" that carries point mutations in its genome segment (Seg) 7 instead of a deletion as in conventional DIPs (cDIPs). Recently, using genetic engineering tools, we generated "OP7 chimera DIPs" that carry point mutations in Seg 7 plus a deletion in Seg 1. Together with cDIPs, OP7 chimera DIPs were produced in shake flasks in the absence of infectious standard virus (STV), rendering UV inactivation unnecessary. However, only part of the virions harvested were OP7 chimera DIPs (78.7%) and total virus titers were relatively low. Here, we describe the establishment of an OP7 chimera DIP production process applicable for large-scale production. To increase total virus titers, we reduced temperature from 37 to 32 °C during virus replication. Production of almost pure OP7 chimera DIP preparations (99.7%) was achieved with a high titer of 3.24 log10(HAU/100 µL). This corresponded to an 11-fold increase relative to the initial process. Next, this process was transferred to a stirred tank bioreactor resulting in comparable yields. Moreover, DIP harvests purified and concentrated by steric exclusion chromatography displayed an increased interfering efficacy in vitro. Finally, a perfusion process with perfusion rate control was established, resulting in a 79-fold increase in total virus yields compared to the original batch process in shake flasks. Again, a very high purity of OP7 chimera DIPs was obtained. This process could thus be an excellent starting point for good manufacturing practice production of DIPs for use as antivirals. Key points: • Scalable cell culture-based process for highly effective antiviral OP7 chimera DIPs • Production of almost pure OP7 chimera DIPs in the absence of infectious virus • Perfusion mode production and purification train results in very high titers [ABSTRACT FROM AUTHOR]

Influenza-associated encephalopathy (IAE) is extremely acute in onset, with high lethality and morbidity within a few days, while the direct pathogenesis by influenza virus in this acute phase in the brain is largely unknown. Here we show that influenza virus enters into the cerebral endothelium and thereby induces IAE. Three-weeks-old young mice were inoculated with influenza A virus (IAV). Physical and neurological scores were recorded and temporal-spatial analyses of histopathology and viral studies were performed up to 72 h post inoculation. Histopathological examinations were also performed using IAE human autopsy brains. Viral infection, proliferation and pathogenesis were analyzed in cell lines of endothelium and astrocyte. The effects of anti-influenza viral drugs were tested in the cell lines and animal models. Upon intravenous inoculation of IAV in mice, the mice developed encephalopathy with brain edema and pathological lesions represented by micro bleeding and injured astrocytic process (clasmatodendrosis) within 72 h. Histologically, massive deposits of viral nucleoprotein were observed as early as 24 h post infection in the brain endothelial cells of mouse models and the IAE patients. IAV inoculated endothelial cell lines showed deposition of viral proteins and provoked cell death, while IAV scarcely amplified. Inhibition of viral transcription and translation suppressed the endothelial cell death and the lethality of mouse models. These data suggest that the onset of encephalopathy should be induced by cerebral endothelial infection with IAV. Thus, IAV entry into the endothelium, and transcription and/or translation of viral RNA, but not viral proliferation, should be the key pathogenesis of IAE. [ABSTRACT FROM AUTHOR]

Influenza A virus pandemics still remain a threat to global health. One class of antiviral drugs, namely, inhibitors of the specific viral enzyme neuraminidase, is predominantly used in the fight against these pandemics. These antivirals include zanamivir (Relenza™) and oseltamivir (Tamiflu™). The viral resistance to this class of compounds steadily increases. The M2 proton channel of influenza A virus is an alternative clinically proven target for antiviral therapy. However, many circulating virus strains bear amino acid mutations in the M2 protein, causing resistance to drugs of the adamantane series, M2 blockers, such as rimantadine and amantadine. Consequently, inhibitors targeting mutants of the M2 channel are urgently needed for public biosafety and health. This review is devoted to structural-functional interactions used in practice and mediated by the action of experimental drugs on the protein target, the transmembrane domain of the influenza virus M2 proton channel. An analysis of the experimental and model structural data available in open access is presented. [ABSTRACT FROM AUTHOR]

Titanium dioxide or aminopropylsilanol nanoparticles were shown to be effective vehicles for delivering oligodeoxyribonucleotides and deoxyribozymes to cells to affect target nucleic acids. In this paper, the proposed principle of the delivery has been implemented in relation to oligoribonucleotides (ORN), components of short interfering RNAs (siRNAs). It has been shown that the obtained ORN-containing nanocomplexes (Si~NH2·ORN) based on aminopropylsilanol nanoparticles penetrate eukaryotic cells. These nanocomplexes have been investigated as agents for suppressing the replication of influenza A virus (IAV) in the cellular system. It has been shown that the ORN strands targeted to (+)RNA and (–)RNA of the IAV 5th segment reduces the titer of the virus by 99.7% and 98.4%, respectively. Thus, oligoribonucleotides in the Si~NH2·ORN nanocomplexes effectively inhibit the replication of the influenza A virus. [ABSTRACT FROM AUTHOR]

Recent reports of a surge in respiratory illnesses among children in Northern China have raised global concern. The Chinese National Health Commission attributes this increase to the lifting of COVID-19 restrictions and the subsequent circulation of known respiratory pathogens such as influenza, respiratory syncytial virus (RSV), Mycoplasma pneumoniae, and SARS-CoV-2. The Chinese Health Authorities have reported an increase in outpatient consultations and hospital admissions for children with M. pneumoniae since May 2023, as well as surges in influenza A/H3N2, adenovirus, and RSV since October 2023. It is important to enhance disease surveillance, maintain immunization, practice social distancing, and promote hand hygiene to reduce the spread of respiratory illnesses. Additionally, antimicrobial stewardship and proactive surveillance are crucial in controlling antimicrobial resistance. [Extracted from the article]

Background Israel confirmed the first case of "flurona"--a co-infection of seasonal flu (IAV) and SARS-CoV-2 in an unvaccinated pregnant woman. This twindemic has been confirmed in multiple countries and underscores the importance of managing respiratory viral illnesses. Results The novel conjugate vaccine was designed by joining four hemagglutinin, three neuraminidase, and four S protein of B-cell epitopes, two hemagglutinin, three neuraminidase, and four S proteins of MHC-I epitopes, and three hemagglutinin, nine neuraminidase, and five S proteins of MHC-II epitopes with linkers and adjuvants. The constructed conjugate vaccine was found stable, non-toxic, non-allergic, and antigenic with 0.6466 scores. The vaccine contained 14.87% alpha helix, 29.85% extended strand, 9.64% beta-turn, and 45.64% random coil, which was modeled to a 3D structure with 94.7% residues in the most favored region of the Ramachandran plot and Z-score of -3.33. The molecular docking of the vaccine with TLR3 represented -1513.9 kcal/mol of binding energy with 39 hydrogen bonds and 514 non-bonded contacts, and 1.582925e-07 of eigenvalue complex. Immune stimulation prediction showed the conjugate vaccine could activate T and B lymphocytes to produce high levels of Th1 cytokines and antibodies. Conclusion The in silico-designed vaccine against IAV and SARS-CoV-2 showed good population coverage and immune response with predicted T- and B-cell epitopes, favorable molecular docking, Ramachandran plot results, and good protein expression. It fulfilled safety criteria, indicating potential for preclinical studies and experimental clinical trials. [ABSTRACT FROM AUTHOR]

Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape. In chickens, influenza A virus relies on host protein ANP32A. Here the authors use CRISPR/Cas9 to generate homozygous gene edited chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. [ABSTRACT FROM AUTHOR]

Human ANP32A and ANP32B are essential but redundant host factors for influenza virus genome replication. While most influenza viruses cannot replicate in edited human cells lacking both ANP32A and ANP32B, some strains exhibit limited growth. Here, we experimentally evolve such an influenza A virus in these edited cells and unexpectedly, after 2 passages, we observe robust viral growth. We find two mutations in different subunits of the influenza polymerase that enable the mutant virus to use a novel host factor, ANP32E, an alternative family member, which is unable to support the wild type polymerase. Both mutations reside in the symmetric dimer interface between two polymerase complexes and reduce polymerase dimerization. These mutations have previously been identified as adapting influenza viruses to mice. Indeed, the evolved virus gains the ability to use suboptimal mouse ANP32 proteins and becomes more virulent in mice. We identify further mutations in the symmetric dimer interface which we predict allow influenza to adapt to use suboptimal ANP32 proteins through a similar mechanism. Overall, our results suggest a balance between asymmetric and symmetric dimers of influenza virus polymerase that is influenced by the interaction between polymerase and ANP32 host proteins. Despite their essentiality, human ANP32A and ANP32B are redundant host factors for influenza virus genome replication. In this work, authors show that an influenza virus grown in cells lacking ANP32A and ANP32B evolved to use ANP32E. They explore the polymerase mutations that enable this, and demonstrate increased virulence in mice. [ABSTRACT FROM AUTHOR]

Abstract—As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation (R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus. [ABSTRACT FROM AUTHOR]

The structure of the full-size M2 channel of influenza virus generated using neural networks and homology modeling was considered. The binding potential of camphecene and ginsamide in the active cavity of the M2 channel filled by amantadine was evaluated. The dynamics of the resulting ligand—protein complexes was simulated. The calculations revealed the presence of ligand—protein contacts stable over the simulation time. The acceptability of existence of complexes of the M2 channel with camphecene and ginsamide in the active site and, as a result, the possibility of a synergistic effect in the inhibition of the influenza virus was hypothesized. [ABSTRACT FROM AUTHOR]

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT. Using a multi-OMICS approach, Haas et al identify 54 human genes and 16 host-targeting chemical compounds that regulate influenza A virus infection in lung epithelial cells, including AHNAK and COBP1 which are also essential for SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Influenza virus is a main cause of acute respiratory tract infections (ARTIs) in children. This is the first double-blind, randomized, and controlled clinical trial examining the efficacy of nasal-spraying probiotic LiveSpo Navax, which contains 5 billion of Bacillus subtilis and B. clausii spores in 5 mL, in supporting treatment of influenza viral infection in pediatric patients. We found that the nasal-spraying Bacillus spores significantly shortened the recovery period and overall treatment by 2 days and increased treatment effectiveness by 58% in resolving all ARTIs' symptoms. At day 2, the concentrations of influenza virus and co-infected bacteria were reduced by 417 and 1152 folds. Additionally, the levels of pro-inflammatory cytokines IL-8, TNF-α, and IL-6 in nasopharyngeal samples were reduced by 1.1, 3.7, and 53.9 folds, respectively. Compared to the standard control group, treatment regimen with LiveSpo Navax demonstrated significantly greater effectiveness, resulting in 26-fold reduction in viral load, 65-fold reduction in bacterial concentration, and 1.1–9.5-fold decrease in cytokine levels. Overall, nasal-spraying Bacillus spores can support the symptomatic treatment of influenza virus-induced ARTIs quickly, efficiently and could be used as a cost-effective supportive treatment for respiratory viral infection in general. Clinical trial registration no: NCT05378022 on 17/05/2022. [ABSTRACT FROM AUTHOR]

The development of a universal influenza vaccine to elicit broad immune responses is essential in reducing disease burden and pandemic impact. In this study, the mosaic vaccine design strategy and genetic algorithms were utilized to optimize the seasonal influenza A virus (H1N1, H3N2) hemagglutinin (HA) and neuraminidase (NA) antigens, which also contain most potential T-cell epitopes. These mosaic immunogens were then expressed as virus-like particles (VLPs) using the baculovirus expression system. The immunogenicity and protection effectiveness of the mosaic VLPs were compared to the commercial quadrivalent inactivated influenza vaccine (QIV) in the mice model. Strong cross-reactive antibody responses were observed in mice following two doses of vaccination with the mosaic VLPs, with HI titers higher than 40 in 15 of 16 tested strains as opposed to limited cross HI antibody levels with QIV vaccination. After a single vaccination, mice also show a stronger level of cross-reactive antibody responses than the QIV. The QIV vaccinations only elicited NI antibodies to a small number of vaccine strains, and not even strong NI antibodies to its corresponding vaccine components. In contrast, the mosaic VLPs caused robust NI antibodies to all tested seasonal influenza virus vaccine strains. Here, we demonstrated the mosaic vaccines induces stronger cross-reactive antibodies and robust more T-cell responses compared to the QIV. The mosaic VLPs also provided protection against challenges with ancestral influenza A viruses of both H1 and H3 subtypes. These findings indicated that the mosaic VLPs were a promising strategy for developing a broad influenza vaccine in future. [ABSTRACT FROM AUTHOR]

Changes of mRNA 3'UTRs by alternative polyadenylation (APA) have been associated to numerous pathologies, but the mechanisms and consequences often remain enigmatic. By combining transcriptomics, proteomics and recombinant viruses we show that all tested strains of IAV, including A/PR/8/34(H1N1) (PR8) and A/Cal/07/2009 (H1N1) (Cal09), cause APA. We mapped the effect to the highly conserved glycine residue at position 184 (G184) of the viral non-structural protein 1 (NS1). Unbiased mass spectrometry-based analyses indicate that NS1 causes APA by perturbing the function of CPSF4 and that this function is unrelated to virus-induced transcriptional shutoff. Accordingly, IAV strain PR8, expressing an NS1 variant with weak CPSF binding, does not induce host shutoff but only APA. However, recombinant IAV (PR8) expressing NS1(G184R) lacks binding to CPSF4 and thereby also the ability to cause APA. Functionally, the impaired ability to induce APA leads to an increased inflammatory cytokine production and an attenuated phenotype in a mouse infection model. Investigating diverse viral infection models showed that APA induction is a frequent ability of many pathogens. Collectively, we propose that targeting of the CPSF complex, leading to widespread alternative polyadenylation of host transcripts, constitutes a general immunevasion mechanism employed by a variety of pathogenic viruses. Here, Bergant et al. provide evidence that Influenza A viruses cause alternative polyadenylation of host mRNAs and abrogation of this function leads to an attenuated phenotype in mice. This may constitute a general immune evasive mechanism employed by a variety of pathogenic viruses. [ABSTRACT FROM AUTHOR]

Since the emergence of COVID-19, several SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) variants have emerged and spread widely. These variants are produced through replication errors of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Seasonal epidemics of influenza are also known to occur because of new variants of influenza A virus (IAV), which are generated by the introduction of mutations by viral RdRp with low fidelity. Variants with different antigenicities appear because of mutations in envelope glycoproteins. In this study, we calculated and compared the mutation rates in genome replication of IAV and SARS-CoV-2. Average mutation rates per passage were 9.01 × 10–5 and 3.76 × 10–6 substitutions/site for IAV and SARS-CoV-2, respectively. The mutation rate of SARS-CoV-2 was 23.9-fold lower than that of IAV because of the proofreading activity of the SARS-CoV-2 RdRp complex. Our data could be useful in establishing effective countermeasures against COVID-19. [ABSTRACT FROM AUTHOR]

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14–15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed. [ABSTRACT FROM AUTHOR]

Background: Influenza is a highly contagious respiratory disease which poses a serious threat to public health globally, causing severe diseases in 3-5 million humans and resulting in 650,000 deaths annually. The current licensed seasonal influenza vaccines lacked cross-reactivity against novel emerging influenza strains as they conferred limited neutralising capabilities. To address the issue, we designed a multi-epitope peptide-based vaccine delivered by the self-adjuvanting PLGA nanoparticles against influenza infections. Methods: A total of six conserved peptides representing B- and T-cell epitopes of Influenza A were identified and they were formulated in either incomplete Freund's adjuvant containing CpG ODN 1826 or being encapsulated in PLGA nanoparticles for the evaluation of immunogenicity in BALB/c mice. Results: The self-adjuvanting PLGA nanoparticles encapsulating the six conserved peptides were capable of eliciting the highest levels of IgG and IFN- γ producing cells. In addition, the immunogenicity of the six peptides encapsulated in PLGA nanoparticles showed greater humoral and cellular mediated immune responses elicited by the mixture of six naked peptides formulated in incomplete Freund's adjuvant containing CpG ODN 1826 in the immunized mice. Peptide 3 from the mixture of six peptides was found to exert necrotic effect on CD3+ T-cells and this finding indicated that peptide 3 should be removed from the nanovaccine formulation. Conclusion: The study demonstrated the self-adjuvanting properties of the PLGA nanoparticles as a delivery system without the need for incorporation of toxic and costly conventional adjuvants in multi-epitope peptide-based vaccines. [ABSTRACT FROM AUTHOR]

Co-infection of respiratory tract viruses and bacteria often result in excess mortality, especially pneumonia caused by influenza viruses and Streptococcus pneumoniae. However, the synergistic mechanisms remain poorly understood. Therefore, it is necessary to develop a clearer understanding of the molecular basis of the interaction between influenza virus and Streptococcus pneumonia. Here, we developed the BALB/c mouse model and the A549 cell model to investigate inflammation and pyroptotic cell death during co-infection. Co-infection significantly activated the NLRP3 inflammasome and induced pyroptotic cell death, correlated with excess mortality. The E3 ubiquitin ligase NEDD4 interacted with both NLRP3 and GSDMD, the executor of pyroptosis. NEDD4 negatively regulated NLRP3 while positively regulating GSDMD, thereby modulating inflammation and pyroptotic cell death. Our findings suggest that NEDD4 may play a crucial role in regulating the GSDMD-mediated pyroptosis signaling pathway. Targeting NEDD4 represents a promising approach to mitigate excess mortality during influenza pandemics by suppressing synergistic inflammation during co-infection of influenza A virus and Streptococcus pneumoniae. [ABSTRACT FROM AUTHOR]

Fever is a common symptom of influenza and coronavirus disease 2019 (COVID-19), yet its physiological role in host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamsters from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who develop moderate I/II disease compared with the minor severity of illness group. These findings implicate a mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner. The ambient environmental temperature changes the extent or severity of a virus infection. Here the authors show that influenza and SARS-CoV-2 infection at higher temperatures promotes gut microbiota derived deoxycholic acid signalling which increases host resistance to infection. [ABSTRACT FROM AUTHOR]

Avian influenza virus (AIV) remains a global threat, with waterfowl serving as the primary reservoir from which viruses spread to other hosts. Highly pathogenic avian influenza (HPAI) H5 viruses continue to be a devastating threat to the poultry industry and an incipient threat to humans. A cross-sectional study was conducted in seven districts of Bangladesh to estimate the prevalence and subtypes (H3, H5, and H9) of AIV in poultry and identify underlying risk factors and phylogenetic analysis of AIVs subtypes H5N1 and H3N8. Cloacal and oropharyngeal swab samples were collected from 500 birds in live bird markets (LBMs) and poultry farms. Each bird was sampled by cloacal and oropharyngeal swabbing, and swabs were pooled for further analysis. Pooled samples were analyzed for the influenza A virus (IAV) matrix (M) gene, followed by H5 and H9 molecular subtyping using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Non-H5 and Non-H9 influenza A virus positive samples were sequenced to identify possible subtypes. Selected H5 positive samples were subjected to hemagglutinin (HA) and neuraminidase (NA) gene sequencing. Multivariable logistic regression was used for risk factor analysis. We found that IAV M gene prevalence was 40.20% (95% CI 35.98–44.57), with 52.38%, 46.96%, and 31.11% detected in chicken, waterfowl, and turkey, respectively. Prevalence of H5, H3, and H9 reached 22%, 3.4%, and 6.9%, respectively. Waterfowl had a higher risk of having AIV (AOR: 4.75), and H5 (AOR: 5.71) compared to chicken; more virus was detected in the winter season than in the summer season (AOR: 4.93); dead birds had a higher risk of AIVs and H5 detection than healthy birds, and the odds of H5 detection increased in LBM. All six H5N1 viruses sequenced were clade 2.3.2.1a-R1 viruses circulating since 2015 in poultry and wild birds in Bangladesh. The 12 H3N8 viruses in our study formed two genetic groups that had more similarity to influenza viruses from wild birds in Mongolia and China than to previous H3N8 viruses from Bangladesh. The findings of this study may be used to modify guidelines on AIV control and prevention to account for the identified risk factors that impact their spread. [ABSTRACT FROM AUTHOR]

Major outbreaks of influenza virus occurred in China in 2017–2018. To describe the pattern of influenza circulation and timing of seasonal epidemics, we analyzed data from influenza-like illness (ILI) specimens on surveillance wards of sentinel hospitals during 2014–2018. Among 1,890,084 ILI cases, 324,211 (17.2%) tested positive for influenza. Influenza A virus (particularly A/H3N2), which circulates annually, was detected in 62% of cases, compared with influenza B virus in 38% of cases. The detection rate of A/H1N1, A/H3N2, B/Victoria, and B/Yamagata viruses were 3.56%, 7.07%, 2.08%, and 3.45%, respectively. Influenza prevalence was generally stable over the four years analyzed, but obvious outbreaks occurred in 2015–2016 (17.28%) and 2017–2018 (22.67%), with B/Victoria and B/Yamagata contributing to these outbreaks, respectively. In the south, a characteristic peak in infections was detected in the summer (week 23–38), which was not detected in the north. Influenza B was found high frequency in school-age children (5–14 years) with 4.78% of B/Victoria and 6.76% of B/Yamagata. Therefore, the epidemiological characteristics of seasonal influenza were complex in China during 2014–2018, presenting distinctions in region, season, and susceptible population. These findings underline the importance of enhancing year-round influenza surveillance and provide a reference for the timing and variety of influenza vaccination. [ABSTRACT FROM AUTHOR]

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement. [ABSTRACT FROM AUTHOR]

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. Here, Bhat et al. show that Influenza A virus mRNAs are exported from the nucleus via the nucleoporin Tpr and the mRNA export complex TREX-2. These mRNAs have low exon number, high mean exon length, and low GC content. A 45-nucleotide RNA signal can mediate export via TREX-2. [ABSTRACT FROM AUTHOR]

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. Key points: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein [ABSTRACT FROM AUTHOR]