Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers.

Tbet+CD11c+ B cells arise during type 1 pathogen challenge, aging, and autoimmunity in mice and humans. Here, we examined the developmental requirements of this B cell subset. In acute infection, T follicular helper (Tfh) cells, but not Th1 cells, drove Tbet+CD11c+ B cell generation through proximal delivery of help. Tbet+CD11c+ B cells developed prior to germinal center (GC) formation, exhibiting phenotypic and transcriptional profiles distinct from GC B cells. Fate tracking revealed that most Tbet+CD11c+ B cells developed independently of GC entry and cell-intrinsic Bcl6 expression. Tbet+CD11c+ and GC B cells exhibited minimal repertoire overlap, indicating distinct developmental pathways. As the infection resolved, Tbet+CD11c+ B cells localized to the marginal zone where splenic retention depended on integrins LFA-1 and VLA-4, forming a competitive memory subset that contributed to antibody production and secondary GC seeding upon rechallenge. Therefore, Tbet+CD11c+ B cells comprise a GC-independent memory subset capable of rapid and robust recall responses.

Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes.

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

Receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection.

NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2(-/-) and Ripk2(-/-) mice are hypersusceptible to infection with influenza A virus. Ripk2(-/-) cells exhibited defective autophagy of mitochondria (mitophagy), leading to enhanced mitochondrial production of superoxide and accumulation of damaged mitochondria, which resulted in greater activation of the NLRP3 inflammasome and production of IL-18. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1(-/-) cells exhibited enhanced mitochondrial production of superoxide and activation of caspase-1. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating activation of the NLRP3 inflammasome and production of IL-18 via ULK1-dependent mitophagy.

Evaluation of the fully automated, sample-to-result Seegene STARlet-AIOS platform for detection of SARS-CoV-2, influenza virus A, influenza virus B, and RSV.

Early, accurate, and bulk detection of respiratory pathogens is essential for patient management and infection control. STARlet-All-in-One System (AIOS) (Seegene) is a new, fully automated, sample-to-result, molecular diagnostic platform. This study describes the first evaluation of STARlet-AIOS, by testing the Allplex™ SARS-CoV-2 (AS) and Allplex™ SARS-CoV-2/FluA/FluB/RSV combination (AC) assays in comparison to the SARS-CoV-2 assays used at our institute. Over a 3-week period, all naso-/oropharyngeal specimens tested for SARS-CoV-2 using either GeneXpert, Panther, or in-house developed test (LDT) were tested on the AIOS using the AS or AC assays. In addition, retrospective cohorts of specimens containing SARS-CoV-2, influenza virus A, influenza virus B, and RSV were tested. Discrepant results were re-tested with another assay used in this study. Hands-on time (HOT) and turn-around time (TAT) of the different systems were monitored and compared. A total of 738 specimens were tested on the AIOS using the AS assay. In addition, 210 specimens were tested using the AC assay. Overall agreement for SARS-CoV-2 detection was established as 98.5% and 95.2% for the AS and AC assay, respectively. Retrospective testing revealed high agreements for all targets, except for influenza virus A (agreement of 87.5%). HOT of the system was comparable to the HOT of GeneXpert and Panther and TAT comparable to Panther and LDT. The AIOS proved to be a robust sample-to-result system with low HOT and moderate TAT. This study showed reliable detection of SARS-CoV-2, influenza virus B, and RSV, whereas detection of influenza virus A using the AC assay appeared to be suboptimal.

IgA potentiates NETosis in response to viral infection.

IgA is the second most abundant antibody present in circulation and is enriched at mucosal surfaces. As such, IgA plays a key role in protection against a variety of mucosal pathogens including viruses. In addition to neutralizing viruses directly, IgA can also stimulate Fc-dependent effector functions via engagement of Fc alpha receptors (Fc-αRI) expressed on the surface of certain immune effector cells. Neutrophils are the most abundant leukocyte, express Fc-αRI, and are often the first to respond to sites of injury and infection. Here, we describe a function for IgA-virus immune complexes (ICs) during viral infections. We show that IgA-virus ICs potentiate NETosis-the programmed cell-death pathway through which neutrophils release neutrophil extracellular traps (NETs). Mechanistically, IgA-virus ICs potentiated a suicidal NETosis pathway via engagement of Fc-αRI on neutrophils through a toll-like receptor-independent, NADPH oxidase complex-dependent pathway. NETs also were capable of trapping and inactivating viruses, consistent with an antiviral function.

Dual-Action Heteromultivalent Glycopolymers Stringently Block and Arrest Influenza A Virus Infection In Vitro and Ex Vivo.

Here, we demonstrate the concerted inhibition of different influenza A virus (IAV) strains using a low-molecular-weight dual-action linear polymer. The 6'-sialyllactose and zanamivir conjugates of linear polyglycerol are optimized for simultaneous targeting of hemagglutinin and neuraminidase on the IAV surface. Independent of IAV subtypes, hemagglutination inhibition data suggest better adsorption of the heteromultivalent polymer than homomultivalent analogs onto the virus surface. Cryo-TEM images imply heteromultivalent compound-mediated virus aggregation. The optimized polymeric nanomaterial inhibits >99.9% propagation of various IAV strains 24 h postinfection in vitro at low nM concentrations and is up to 10000× more effective than the commercial zanamivir drug. In a human lung ex vivo multicyclic infection setup, the heteromultivalent polymer outperforms the commercial drug zanamivir and homomultivalent analogs or their physical mixtures. This study authenticates the translational potential of the dual-action targeting approach using small polymers for broad and high antiviral efficacy.

Decline in intravenous immunoglobulin titer against influenza virus A/H2N2 derived from donors in Japan.

Human influenza A/H2N2 can induce a pandemic in the future. This study evaluated the hemagglutination inhibition and neutralizing titers of intravenous immunoglobulin against A/H2N2 viruses, indicating the status of the donor population. In this study, the antibody titers decreased during the study period-2012-2021-suggesting a reduction in the immunity of the studied population.

Decreased expression of surfactant Protein-C and CD74 in alveolar epithelial cells during influenza virus A(H1N1)pdm09 and H3N2 infection.

The primary replication site of Influenza A virus (IAV) is type II alveolar epithelial cells (AECII), which are central to normal lung function and present important immune functions. Surfactant components are synthesized primarily by AECII, which play a crucial role in host defense against infection. The aim of this study was to analyze if the impact of influenza infection is differential between A(H1N1)pdm09 and A/Victoria/3/75 (H3N2) on costimulatory molecules and ProSP-C expression in AECII from BALB/c mice infected and A549 cell line infected with both strains. Pandemic A(H1N1)pdm09 and A/Victoria/3/75 (H3N2) were used to infect BALB/c mice and the A549 cell line. We evaluated the surface expression of co-stimulatory molecules (CD45/CD31/CD74/ProSP-C) in AECII and A549 cell lines. Our results showed a significant decrease in ProSP-C+ CD31- CD45- and CD74+ CD31- CD45- expression in AECII and A549 cell line with the virus strain A(H1N1)pdm09 versus A/Victoria/3/75 (H3N2) and controls (non-infection conditions). Our findings indicate that changes in the expression of ProSP-C in AECII and A549 cell lines in infection conditions could result in dysfunction leading to decreased lung compliance, increased work of breathing and increased susceptibility to injury.

Severe influenza virus infection in children admitted to the PICU: Comparison of influenza A and influenza B virus infection.

Although the influenza virus usually causes a self-limiting disease, deaths are reported even in children without risk factors. We aimed to identify the clinical features, mortality associated with severe influenza A and B virus infections of children admitted to the pediatric intensive care unit (PICU). We conducted a retrospective study of children with confirmed influenza infection between 2012 and 2019 who were admitted to the PICU. Demographic features, risk factors, clinical data, microbiological data, complications, and outcomes were collected. Over seven influenza seasons (2012-2011 to 2015-2016), 713 children diagnosed with laboratory-confirmed influenza-related LRTI, and PICU admission was needed in 6% (46/713) of the patients. Thirty-one patients (67.4%) were diagnosed with influenza A and 15 patients were diagnosed with influenza B. Epidemiologic and clinical characteristics were similar in both influenza types, lactate dehydrogenase levels were significantly higher for influenza A than for influenza B infections. Although the influenza A to B ratio among the patients admitted to the PICU was 2.06, the percentage of cases requiring PICU admission was nearly two times higher in influenza B cases. There was no statistically significant difference in disease severity and complications in patients with influenza A and influenza B.

Comparison of proteins with anti-influenza virus effects in parotid and submandibular-sublingual saliva in humans.

BACKGROUND: Saliva possesses antiviral activity, with submandibular-sublingual (SMSL) saliva having higher antiviral activity than parotid saliva. Various salivary proteins have inactivating effects on influenza A virus (IAV), but the detailed relationship between antiviral proteins and salivary anti-IAV activities in the parotid and SMSL glands is unknown. Here, to identify salivary proteins with anti-IAV activity, salivary proteins from parotid and SMSL glands were identified, quantified, and compared using liquid chromatography-mass spectrometry. METHODS: Twelve healthy male volunteers participated in the study. Parotid and SMSL saliva was collected by suction and collection devices. We assessed anti-IAV activities, protein concentrations, and protein-bound sialic acid concentrations in parotid and SMSL saliva. RESULTS: SMSL had significantly higher anti-IAV activity than parotid saliva. SMSL also had higher concentrations of glycoproteins, such as mucin 5B and mucin 7, protein-bound sialic acid, cystatins, and lysozyme C, compared with parotid saliva. Salivary mucin 5B and mucin 7 concentrations significantly positively correlated with the salivary protein-bound sialic acid concentration. Salivary anti-IAV activity significantly positively correlated with protein-bound sialic acid, mucin 5B, mucin 7, cystatin-C, -S, and -SN concentrations. CONCLUSION: Salivary mucins, cystatins, and lysozyme C contribute to the high anti-IAV activity of SMSL saliva.

Variable Sensitivity of SARS-CoV-2 Molecular Detection in European Expert Laboratories: External Quality Assessment, June and July 2020.

During the ongoing coronavirus disease 2019 (COVID-19) outbreak, robust detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key element for clinical management and to interrupt transmission chains. We organized an external quality assessment (EQA) of molecular detection of SARS-CoV-2 for European expert laboratories. An EQA panel composed of 12 samples, containing either SARS-CoV-2 at different concentrations to evaluate sensitivity or other respiratory viruses to evaluate specificity of SARS-CoV-2 testing, was distributed to 68 laboratories in 35 countries. Specificity samples included seasonal human coronaviruses hCoV-229E, hCoV-NL63, and hCoV-OC43, as well as Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and human influenza viruses A and B. Sensitivity results differed among laboratories, particularly for low-concentration SARS-CoV-2 samples. Results indicated that performance was mostly independent of the selection of specific extraction or PCR methods.

Baloxavir treatment of oseltamivir-resistant influenza A/H1pdm09 in two immunocompromised patients.

Few treatment options are available for oseltamivir-resistant influenza. It has been proposed that baloxavir can be effective in this setting due to its distinct mechanism of action but clinical experience is lacking for immunocompromised patients. We report two such cases treated with baloxavir after failure of oseltamivir and detection of oseltamivir resistance mutations. Baloxavir/zanamivir combination therapy was effective in one patient, but persistent viral shedding was noted with baloxavir monotherapy in the other patient.

Chemoenzymatic Total Synthesis of Sorbicatechol Structural Analogues and Evaluation of Their Antiviral Potential.

Sorbicillinoids are fungal polyketides characterized by highly complex and diverse molecular structures, with considerable stereochemical intricacy combined with a high degree of oxygenation. Many sorbicillinoids possess promising biological activities. An interesting member of this natural product family is sorbicatechol A, which is reported to have antiviral activity, particularly against influenza A virus (H1N1). Through a straightforward, one-pot chemoenzymatic approach with recently developed oxidoreductase SorbC, the characteristic bicyclo[2.2.2]octane core of sorbicatechol is structurally diversified by variation of its natural 2-methoxyphenol substituent. This facilitates the preparation of a focused library of structural analogues bearing substituted aromatic systems, alkanes, heterocycles, and ethers. Fast access to this structural diversity provides an opportunity to explore the antiviral potential of the sorbicatechol family.

Inside the lungs of COVID-19 disease.

In the setting of the coronavirus disease 2019 (COVID-19) pandemic, only few data regarding lung pathology induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is available, especially without medical intervention interfering with the natural evolution of the disease. We present here the first case of forensic autopsy of a COVID-19 fatality occurring in a young woman, in the community. Diagnosis was made at necropsy and lung histology showed diffuse alveolar damage, edema, and interstitial pneumonia with a geographically heterogeneous pattern, mostly affecting the central part of the lungs. This death related to COVID-19 pathology highlights the heterogeneity and severity of central lung lesions after natural evolution of the disease.

Comparative thermostability analysis of zoonotic and human influenza virus A and B neuraminidase.

Neuraminidase (NA) thermostability of influenza A and B viruses isolated from birds, swine and humans was measured to evaluate its variability associated with host body temperature. The highest 50% inactivation temperature (IT50) was observed with H3N8 avian influenza virus (74 °C), and the lowest IT50 was observed with the seasonal human H3N2 virus (45.5 °C). The IT50 values of A(H1N1)pdm09 viruses 56.4-58.5 °C were statistically higher than that of the prepandemic strain A/Solomon Islands/03/06 (52.5 °C). An analysis of Ca2+ binding sites revealed the correspondence of amino acid changes to NA thermostability. This study demonstrates that changes in NA thermostability correspond to differences in host body temperature.

Energetic cost of building a virus.

Viruses are incapable of autonomous energy production. Although many experimental studies make it clear that viruses are parasitic entities that hijack the molecular resources of the host, a detailed estimate for the energetic cost of viral synthesis is largely lacking. To quantify the energetic cost of viruses to their hosts, we enumerated the costs associated with two very distinct but representative DNA and RNA viruses, namely, T4 and influenza. We found that, for these viruses, translation of viral proteins is the most energetically expensive process. Interestingly, the costs of building a T4 phage and a single influenza virus are nearly the same. Due to influenza's higher burst size, however, the overall cost of a T4 phage infection is only 2-3% of the cost of an influenza infection. The costs of these infections relative to their host's estimated energy budget during the infection reveal that a T4 infection consumes about a third of its host's energy budget, whereas an influenza infection consumes only ≈ 1%. Building on our estimates for T4, we show how the energetic costs of double-stranded DNA phages scale with the capsid size, revealing that the dominant cost of building a virus can switch from translation to genome replication above a critical size. Last, using our predictions for the energetic cost of viruses, we provide estimates for the strengths of selection and genetic drift acting on newly incorporated genetic elements in viral genomes, under conditions of energy limitation.

Influenza infection triggers disease in a genetic model of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Most MS patients experience periods of symptom exacerbation (relapses) followed by periods of partial recovery (remission). Interestingly, upper-respiratory viral infections increase the risk for relapse. Here, we used an autoimmune-prone T-cell receptor transgenic mouse (2D2) and a mouse-adapted human influenza virus to test the hypothesis that upper-respiratory viral infection can cause glial activation, promote immune cell trafficking to the CNS, and trigger disease. Specifically, we inoculated 2D2 mice with influenza A virus (Puerto Rico/8/34; PR8) and then monitored them for symptoms of inflammatory demyelination. Clinical and histological experimental autoimmune encephalomyelitis was observed in ∼29% of infected 2D2 mice. To further understand how peripheral infection could contribute to disease onset, we inoculated wild-type C57BL/6 mice and measured transcriptomic alterations occurring in the cerebellum and spinal cord and monitored immune cell surveillance of the CNS by flow cytometry. Infection caused temporal alterations in the transcriptome of both the cerebellum and spinal cord that was consistent with glial activation and increased T-cell, monocyte, and neutrophil trafficking to the brain at day 8 post infection. Finally, Cxcl5 expression was up-regulated in the brains of influenza-infected mice and was elevated in cerebrospinal fluid of MS patients during relapse compared with specimens acquired during remission. Collectively, these data identify a mechanism by which peripheral infection may exacerbate MS as well as other neurological diseases.

Viral infection and pediatric pancreatitis.

We have read the article entitled "A rare association: acute pancreatitis caused by the influenza virus A with secondary appendicitis in a six-year-old girl" by Láinez Ramos-Bossini AJ et al. with great interest. This case report is successful and informative. We are specifically interested in viruses and pediatric pancreatitis and would like to mention a few crucial points about pediatric pancreatitis caused by a viral infection.

An investigation into respiratory tract viruses in children with acute lower respiratory tract infection or wheezing.

BACKGROUND: This study aimed to determine the frequencies of respiratory tract viruses in patient (acute lower respiratory tract infection [LRTI] or wheezing) and control (history of asthma without symptoms) groups. METHODS: Using multiplex-polymerase chain reaction (PCR), respiratory tract viruses were investigated in the respiratory tract specimens from patient and control groups followed in the Pediatric Clinic. RESULTS: The viruses detected in the patient and control groups (P=0.013) were as follows, respectively: rhinoviruses A, B, C (25.6% and 36.7%), influenza virus A (21.1% and 0.0%), parainfluenza virus type 1 (7.8% and 1.7%), parainfluenza virus type 4 (5.6% and 0.0%), adenoviruses A, B, C, D, E (4.4% and 1.7%), parainfluenza virus type 3 (4.4% and 1.7%), coronaviruses 229E and NL63 (4.4% and 1.7%), coronavirus OC43 (3.3% and 0.0%), respiratory syncytial virus A (3.3% and 0.0%), parainfluenza virus type 2 (2.2% and 0.0%), influenza virus B (2.2% and 0.0%), and respiratory syncytial virus B (1.1% and 1.7%). No bocavirus, metapneumovirus or enterovirus was found in any specimen. Statistically significant differences in the detection of influenza virus A (P=0.000), the total detection of parainfluenza viruses (P=0.008) and coinfection (P=0.004) were observed between the patient and control groups. CONCLUSIONS: The advantage of our study compared with other studies is the inclusion of not only wheezing patients but also children with asthma without symptom. The higher detection of rhinoviruses both in patient and control groups give rise to thought that these viruses may be responsible for asthma exacerbations and may be related with long duration of virus shedding.

A rare association: acute pancreatitis caused by the influenzavirus A with secondary appendicitis in a six-year-old girl.

Acute pancreatitis (AP) is the most frequent pancreatic disorder in children and abdominal ultrasound (AUS) is very helpful in the clinical management. Even though several agents have been implicated in the etiology of AP, influenza virus A (IVA) is exceptional. We report the case of a 6-year-old girl who presented with generalized abdominal pain and flu-like symptoms. Blood tests and AUS revealed typical findings of AP and a nasopharyngeal aspirate was positive for IVA. Twenty-four hours later, the patient developed signs of acute appendicitis, which was also confirmed by AUS. This case highlights the importance of AUS in the management of acute abdominal conditions in children, including reactive entities such as appendicitis, as well as the need to consider IVA as a potential causal agent of AP.