mRNA-encoded Cas13 treatment of Influenza via site-specific degradation of genomic RNA.

The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections.

Innovations in cell culture-based influenza vaccine manufacturing - from static cultures to high cell density cultivations.

Influenza remains a serious global health concern, causing significant morbidity and mortality each year. Vaccination is crucial to mitigate its impact, but requires rapid and efficient manufacturing strategies to handle timing and supply. Traditionally relying on egg-based production, the field has witnessed a paradigm shift toward cell culture-based methods offering enhanced flexibility, scalability, and process safety. This review provides a concise overview of available cell substrates and technological advancements. We summarize crucial steps toward process intensification - from roller bottle production to dynamic cultures on carriers and from suspension cultures in batch mode to high cell density perfusion using various cell retention devices. Moreover, we compare single-use and conventional systems and address challenges including defective interfering particles. Taken together, we describe the current state-of-the-art in cell culture-based influenza virus production to sustainably meet vaccine demands, guarantee a timely supply, and keep up with the challenges of seasonal epidemics and global pandemics.

Associations of proton pump inhibitors with susceptibility to influenza, pneumonia, and COVID-19: Evidence from a large population-based cohort study.

Background: Adverse effects of proton pump inhibitors (PPIs) have raised wide concerns. The association of PPIs with influenza is unexplored, while that with pneumonia or COVID-19 remains controversial. Our study aims to evaluate whether PPI use increases the risks of these respiratory infections. Methods: The current study included 160,923 eligible participants at baseline who completed questionnaires on medication use, which included PPI or histamine-2 receptor antagonist (H2RA), from the UK Biobank. Cox proportional hazards regression and propensity score-matching analyses were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs). Results: Comparisons with H2RA users were tested. PPI use was associated with increased risks of developing influenza (HR 1.32, 95% CI 1.12-1.56) and pneumonia (hazard ratio [HR] 1.42, 95% confidence interval [CI] 1.26-1.59). In contrast, the risk of COVID-19 infection was not significant with regular PPI use (HR 1.08, 95% CI 0.99-1.17), while the risks of severe COVID-19 (HR 1.19. 95% CI 1.11-1.27) and mortality (HR 1.37. 95% CI 1.29-1.46) were increased. However, when compared with H2RA users, PPI users were associated with a higher risk of influenza (HR 1.74, 95% CI 1.19-2.54), but the risks with pneumonia or COVID-19-related outcomes were not evident. Conclusions: PPI users are associated with increased risks of influenza, pneumonia, as well as COVID-19 severity and mortality compared to non-users, while the effects on pneumonia or COVID-19-related outcomes under PPI use were attenuated when compared to the use of H2RAs. Appropriate use of PPIs based on comprehensive evaluation is required. Funding: This work is supported by the National Natural Science Foundation of China (82171698, 82170561, 81300279, 81741067, 82100238), the Program for High-level Foreign Expert Introduction of China (G2022030047L), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2021B1515020003), the Guangdong Basic and Applied Basic Research Foundation (2022A1515012081), the Foreign Distinguished Teacher Program of Guangdong Science and Technology Department (KD0120220129), the Climbing Program of Introduced Talents and High-level Hospital Construction Project of Guangdong Provincial People's Hospital (DFJH201923, DFJH201803, KJ012019099, KJ012021143, KY012021183), and in part by VA Clinical Merit and ASGE clinical research funds (FWL).

Seasonal influenza in children: complications, treatment, prevention (subject publication review).

OBJECTIVE: Aim: To study the specificity of seasonal flu in children, in particular, in young children, as well as treatment, prevention and complications of seasonal flu. PATIENTS AND METHODS: Materials and Methods: For the methodological justification of the article, we used the pool of research technologies. Methods of theoretical analysis, system-analytical, comparative methods provided us with the opportunity to characterize the features of influenza incidence in children. CONCLUSION: Conclusions: A distinctive feature of influenza is the high lability of the genes of the infectious agent. In this regard, it is extremely important to timely update information about new strains of the pathogen, creation of new types of vaccines and antiviral drugs, as well as changes in the course of the disease. Our literature review is intended to improve the medical community.

INfluenza VaccInation To mitigate typE 1 Diabetes (INVITED): a study protocol for a randomised, double-blind, placebo-controlled clinical trial in children and adolescents with recent-onset type 1 diabetes.

INTRODUCTION: Children and adolescents with recent-onset type 1 diabetes (T1D) commonly maintain a certain level of insulin production during the remission phase, which can last months to years. Preserving ß-cell function can reduce T1D complications and improve glycaemic control. Influenza vaccination has pleiotropic effects and administration of the vaccine during the early phases of T1D may offer ß-cell protection. This study aims to assess the effect of influenza vaccination on preserving ß-cell function in children and adolescents with recent-onset T1D. METHODS AND ANALYSIS: The INfluenza VaccInation To mitigate typE 1 Diabetes trial is a randomised, double-blind, placebo-controlled, multicentre trial in paediatric patients with recent-onset T1D aged 7-17 years. 100 participants will be randomised in a 1:1 ratio to receive either a standard inactivated quadrivalent influenza vaccine or a placebo within 14 days of diagnosis. The primary outcome is a difference in mean change (from baseline to 12 months) in C-peptide level between groups during a 2-hour mixed-meal tolerance test. Secondary outcomes include mean change (from baseline to 6 months) in C-peptide levels, haemoglobin A1c, ambulatory glucose profiles and insulin requirements. Exploratory outcomes are diabetes-related autoantibodies, inflammatory markers and serum haemagglutinin inhibition antibody titres against the influenza viruses. The current treatment for T1D is largely symptomatic, relying on insulin administration. There is a pressing need for novel pharmacological approaches aimed at modulating the immune system to preserve residual ß-cell function. Existing immunotherapies are cost-prohibitive and associated with multiple side effects, whereas influenza vaccination is inexpensive and generally well tolerated. A positive outcome of this study holds potential for immediate implementation into standard care for children and adolescents with recent-onset T1D and may guide future research on immune modulation in T1D. ETHICS AND DISSEMINATION: Ethical approval was obtained from Danish Health Authorities prior to participant enrollment. The trial results will be submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT05585983 and EudraCT Number 2022-500906-17-01.

Binary and quaternary mixtures of perfluoroalkyl substances (PFAS) differentially affect the immune response to influenza A virus infection.

Per- and polyfluoroalkyl substances (PFAS) are anthropogenic organofluorine compounds that persist indefinitely in the environment and bioaccumulate throughout all trophic levels. Biomonitoring efforts have detected multiple PFAS in the serum of most people. Immune suppression has been among the most consistent effects of exposure to PFAS. PFAS often co-occur as mixtures in the environment, however, few studies have examined immunosuppression of PFAS mixtures or determined whether PFAS exposure affects immune function in the context of infection. In this study, mixtures containing two or four different PFAS and a mouse model of infection with influenza A virus (IAV) were used to assess immunotoxicity of PFAS mixtures. PFAS were administered via the drinking water as either a binary mixture of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) or quaternary mixture of PFOS, PFOA, perfluorohexane sulfonate (PFHxS), and perfluorononanoic acid (PFNA). The results indicated that the binary mixture affected the T-cell response, while the quaternary mixture affected the B-cell response to infection. These findings indicate that the immunomodulatory effects of PFAS mixtures are not simply additive, and that the sensitivity of immune responses to PFAS varies by cell type and mixture. The study also demonstrates the importance of studying adverse health effects of PFAS mixtures.

Association Between Influenza Vaccination and SARS-CoV-2 Infection.

BACKGROUND: Recent data in nonpregnant individuals suggest a protective effect of influenza vaccination against SARS-CoV-2 infection and its severity. OBJECTIVES: Our primary objective was to evaluate whether influenza vaccination was associated with COVID-19 severity and pregnancy and neonatal outcomes among those infected with SARS-CoV-2. The secondary objective was to examine the association between influenza vaccination and SARS-CoV-2 infection. STUDY DESIGN: Secondary analysis of a multicenter retrospective cohort of pregnant people who tested positive for SARS-CoV-2 between March and August 2020, and a cohort of random deliveries during the same time period. The associations between 2019 influenza vaccination and the primary outcome of moderate-to-critical COVID-19 as well as maternal and perinatal outcomes were examined among all people who tested positive for SARS-CoV-2 between March and August 2020. The association between 2019 influenza vaccination and having a positive SARS-CoV-2 test was examined among a cohort of individuals who delivered on randomly selected dates between March and August 2020. Univariable and multivariable analyses were performed. RESULTS: Of 2325 people who tested positive for SARS-CoV-2, 1068 (45.9%) were vaccinated against influenza in 2019. Those who received the influenza vaccine were older, leaner, more likely to have private insurance, and identify as White or Hispanic. They were less likely to smoke tobacco and identify as Black. Overall, 419 (18.0%) had moderate, 193 (8.3%) severe, and 52 (2.2%) critical COVID-19. There was no association between influenza vaccination and moderate-to-critical COVID-19 (29.2% vs. 28.0%, adjusted OR 1.10, 95% CI 0.90-1.34) or adverse maternal and perinatal outcomes among those who tested positive. Of 8152 people who delivered in 2020, 4658 (57.1%) received the influenza vaccine. Prior vaccination was not associated with a difference in the odds of SARS-CoV-2 infection (3.8% vs. 4.2%, adjusted OR 0.94, 95% CI 0.74-1.19). CONCLUSION: Prior influenza vaccination was not associated with decreased severity of COVID-19 or lower odds of SARS-CoV-2 infection in pregnancy.

Surveillance of respiratory viruses by aerosol screening in indoor air as an early warning system for epidemics.

The development of effective methods for the surveillance of seasonal respiratory viruses is required for the timely management of outbreaks. We aimed to survey Influenza-A, Influenza-B, RSV-A, Rhinovirus and SARS-CoV-2 surveillance in a tertiary hospital and a campus over 5 months. The effectiveness of air screening as an early warning system for respiratory viruses was evaluated in correlation with respiratory tract panel test results. The overall viral positivity was higher on the campus than in the hospital (55.0% vs. 38.0%). Influenza A was the most prevalent pathogen in both locations. There were two influenza peaks (42nd and 49th weeks) in the hospital air, and a delayed peak was detected on campus in the 1st-week of January. Panel tests indicated a high rate of Influenza A in late December. RSV-A-positivity was higher on the campus than the hospital (21.6% vs. 7.4%). Moreover, we detected two RSV-A peaks in the campus air (48th and 51st weeks) but only one peak in the hospital and panel tests (week 49). Although rhinovirus was the most common pathogen in panel tests, rhinovirus positivity was low in air samples. The air screening for Influenza-B and SARS-Cov-2 revealed comparable positivity rates with panel tests. Air screening can be integrated into surveillance programs to support infection control programs for potential epidemics of respiratory virus infections except for rhinoviruses.

Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses.

Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01µM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.

Influenza Cerebellitis in a 3-Year-Old Male.

Acute cerebellitis (AC) is often a para-infectious inflammatory process that usually presents with a variable clinical picture; often with fever, nausea, headache, altered mental status, gait abnormalities, and coordination defects. It is usually a complication of an infectious process or as a result of post-infectious autoimmune mechanisms. We report a case of a 3 year old male with influenza A who presented with an acute encephalitic picture whose course and radiologic studies demonstrate cerebellar changes strongly compatible with AC.

Influenza A viral burst size from thousands of infected single cells using droplet quantitative PCR (dqPCR).

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses.

Increased adherence to influenza vaccination among Palermo family pediatricians: a study on safety and compliance of qLAIV vaccination.

BACKGROUND: Influenza represents a serious public health threat, especially for the management of severe cases and complications of the disease, requiring the implementation of control measures. We aimed to assess the acceptance and impact of qLAIV vaccination among a representative sample of family paediatricians (FPs) operating in Palermo Local Health Authority (LHA). To this end we evaluated vaccination coverage rates, comparing it with that observed in Sicilian context, while actively monitoring possible adverse reactions and their severity. METHODS: An observational descriptive non-controlled study was conducted in two phases, from September 2022 to June 2023. The first phase involved a formative and educational intervention with a pre-intervention questionnaire to assess the knowledge and attitudes of FPs on paediatric influenza vaccination. The second phase consisted of an active surveillance on qLAIV safety and acceptance among the paediatric population assisted by the participating FPs, from October 2022 to April 2023. Frequencies, chi-squared tests, and comparisons statistics were performed using Stata/MP 14.1. RESULTS: The overall coverage rate among the paediatric population involved in the intervention was 13.2%, with an I.M./qLAIV ratio of vaccine administered of 1/4.25. This coverage rate was significantly higher (p-value <0.001) when compared to the average values reported in the population under the Palermo Local Health Authority (LHA) (6.7%) and in the entire Sicily (5.9%). Adverse events in the qLAIV group were mild, with only 3.3% experiencing them, primarily presenting as a feverish rise (3.2%). No severe adverse reaction was reported. CONCLUSIONS: The educational intervention significantly raised paediatric influenza vaccination rates among the participating FPs, and in general improved influenza vaccination coverage rates in the Palermo's LHU. Minimal, non-serious adverse events underscored the vaccine's safety. Training sessions ensured paediatricians stayed informed, enabling them to provide comprehensive information to parents for secure and informed vaccination decisions in their practices.

Opening another door on influenza entry.

In this issue of Cell Host & Microbe, Karakus et al. find that an influenza virus enters cells by exclusively binding to a protein instead of sugars.

Anti-Influenza A Potential of Tagetes erecta Linn. Extract Based on Bioinformatics Analysis and In Vitro Assays.

Tagetes erecta Linn. (TE) is traditionally used to treat cardiovascular, renal, and gastrointestinal diseases. In this study, we investigated the active compounds and targets of TE extract that may exert antiviral effects against influenza A. Active compounds and targets of TE extract were identified using the Traditional Chinese Medicine Systems Pharmacology database (TCSMP). The influenza A-related gene set was screened using GeneCards and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A protein-protein interaction (PPI) network was built to establish the hub targets. Pathway and target studies were conducted using Gene Expression Omnibus (GEO). The interactions between active compounds and potential targets were assessed by molecular docking. An in vitro study was performed using antiviral and plaque reduction assays. From the compound and target search, we identified 6 active compounds and 95 potential targets. We retrieved 887 influenza-associated target genes and determined 14 intersecting core targets between TE and influenza. After constructing a compound-target network, we discovered lutein and beta-carotene to be the key compounds. Next, PPI network analysis identified the top three hub genes associated with influenza (IL-6, HIF1A, and IL-1ß). Similarly, GEO analysis revealed IL-6, TGFB1, and CXCL8 to be the top three target genes. In our docking study, we identified that lutein and IL-6 had the strongest bindings. Our in vitro experimental results revealed that the TE extract exhibited therapeutic rather than prophylactic effects on influenza disease. We identified lutein as a main active compound in TE extract, and IL-6 as an important target associated with influenza, by using data mining and bioinformatics. Our in vitro findings indicated that TE extract exerted protective properties against the influenza A virus. We speculated that lutein, as a key active component in TE extract, is largely responsible for its antiviral effects. Therefore, we suggest TE extract as an alternative in the treatment of influenza.

miR-126a-5p inhibits H1N1-induced inflammation and matrix protease secretion in lung fibroblasts by targeting ADAMTS-4.

Upregulation of ADAMTS-4 has been reported to have an important role in lung injury, and ADAMTS-4 expression is regulated by miR-126a-5p in abdominal aortic aneurysms. The aim of this study was to investigate whether miR-126a-5p/ADAMTS-4 plays a role in influenza-virus-induced lung injury. Lung fibroblasts were infected with H1N1 influenza virus to detect changes in miR-126a-5p and ADAMTS-4 expression, and cell viability was measured by CCK-8 assay. Inflammatory factors and matrix protease levels were examined using ELISA kits, and cell apoptosis was assessed by measuring the levels of apoptosis-related proteins. A dual luciferase assay was used to verify the regulatory relationship between miR-126a-5p and ADAMTS-4. H1N1 influenza virus reduced fibroblast viability, inhibited miR-126a-5p expression, and promoted ADAMTS-4 expression. Overexpression of miR-126a-5p attenuated the cellular inflammatory response, apoptosis, matrix protease secretion, and virus replication. Luciferase reporter assays revealed that miR-126a-5p inhibited ADAMTS-4 expression by targeting ADAMTS-4 mRNA. Further experiments showed that overexpression of ADAMTS-4 significantly reversed the inhibitory effects of miR-126a-5p on fibroblast inflammation, apoptosis, matrix protease secretion, and virus replication. Upregulation of miR-126a-5p inhibits H1N1-induced apoptosis, inflammatory factors, and matrix protease secretion, as well as virus replication in lung fibroblasts.

Tropism for ciliated cells is the dominant driver of influenza viral burst size in the human airway.

Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro- and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types.