SARS-CoV-2 Omicron variant replication in human bronchus and lung ex vivo.

The emergence of SARS-CoV-2 variants of concern with progressively increased transmissibility between humans is a threat to global public health. The Omicron variant of SARS-CoV-2 also evades immunity from natural infection or vaccines1, but it is unclear whether its exceptional transmissibility is due to immune evasion or intrinsic virological properties. Here we compared the replication competence and cellular tropism of the wild-type virus and the D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants in ex vivo explant cultures of human bronchi and lungs. We also evaluated the dependence on TMPRSS2 and cathepsins for infection. We show that Omicron replicates faster than all other SARS-CoV-2 variants studied in the bronchi but less efficiently in the lung parenchyma. All variants of concern have similar cellular tropism compared to the wild type. Omicron is more dependent on cathepsins than the other variants of concern tested, suggesting that the Omicron variant enters cells through a different route compared with the other variants. The lower replication competence of Omicron in the human lungs may explain the reduced severity of Omicron that is now being reported in epidemiological studies, although determinants of severity are multifactorial. These findings provide important biological correlates to previous epidemiological observations.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Replication of Novel Zoonotic-Like Influenza A(H3N8) Virus in Ex Vivo Human Bronchus and Lung.

Human infection with avian influenza A(H3N8) virus is uncommon but can lead to acute respiratory distress syndrome. In explant cultures of the human bronchus and lung, novel H3N8 virus showed limited replication efficiency in bronchial and lung tissue but had a higher replication than avian H3N8 virus in lung tissue.

SGLT2 Inhibitors: The Next Blockbuster Multifaceted Drug?

Sodium glucose cotransporter 2 inhibitor (SGLT2i) is a class of drugs that were originally intended for decreasing blood glucose in diabetes. However, recent trials have shown that there are other beneficial effects. Major clinical trials involving SGLT2i medications from 2015 to 2022 were reviewed using PUBMED search. Recent major SGLT2i landmark trials have demonstrated benefits for cardiovascular disease (reduce major adverse cardiovascular events (heart attack, stroke, cardiovascular death), hospitalization for heart failure, all-cause death), and renal disease (delay the onset of dialysis) regardless of diabetic status. The consistent cardiorenal benefits observed in major landmark trials have resulted in the rapid adoption of SGLT2i therapy not only in diabetes guidelines but also cardiovascular and renal guidelines.

Cooperativity in Shape-Persistent Bis-(Zn-salphen) Catalysts for Efficient Cyclic Carbonate Synthesis under Mild Conditions.

A series of conformationally rigid (Zn-salphen)2 complexes with a planar bridging component (xanthene or dibenzofuran) are described. Conformational changes for these assemblies are essentially limited to the axial rotation of the Zn-salphen moieties; however, such geometric constraints crucially permit the subtle tuning of the intermetallic separation and geometry to potentially enhance catalytic activity (and cooperative effects). The complexes have been investigated as catalysts in conjunction with nBu4NI for the coupling of CO2 with epoxides. Selected dibenzofuran derivatives are significantly more active for the production of cyclic carbonate than their mononuclear analogues under identical conditions and concentrations of Zn sites. High initial turnover frequencies (up to 29 000 h-1; 14 500 h-1 per Zn, using 10 bar of CO2 at 95 °C) and excellent efficiencies under mild conditions (1 bar of CO2 at 55 °C) have been achieved. Kinetic studies using in situ (ReactIR) spectroscopy and density functional theory calculations have been performed, which reveal the existence of an intramolecular rate component and a preference for the cooperative pathway as well as transition states that depict the Zn sites operating in tandem. Taken together, these results provide strong evidence of cooperative reactivity in these Zn2 catalysts.

Tropism of SARS-CoV-2, SARS-CoV, and Influenza Virus in Canine Tissue Explants.

BACKGROUND: Human spillovers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to dogs and the emergence of a highly contagious avian-origin H3N2 canine influenza virus have raised concerns on the role of dogs in the spread of SARS-CoV-2 and their susceptibility to existing human and avian influenza viruses, which might result in further reassortment. METHODS: We systematically studied the replication kinetics of SARS-CoV-2, SARS-CoV, influenza A viruses of H1, H3, H5, H7, and H9 subtypes, and influenza B viruses of Yamagata-like and Victoria-like lineages in ex vivo canine nasal cavity, soft palate, trachea, and lung tissue explant cultures and examined ACE2 and sialic acid (SA) receptor distribution in these tissues. RESULTS: There was limited productive replication of SARS-CoV-2 in canine nasal cavity and SARS-CoV in canine nasal cavity, soft palate, and lung, with unexpectedly high ACE2 levels in canine nasal cavity and soft palate. Canine tissues were susceptible to a wide range of human and avian influenza viruses, which matched with the abundance of both human and avian SA receptors. CONCLUSIONS: Existence of suitable receptors and tropism for the same tissue foster virus adaptation and reassortment. Continuous surveillance in dog populations should be conducted given the many chances for spillover during outbreaks.

Saccharide-Functionalized Poly(Zn-salphen)-alt-(m- and p-phenyleneethynylene)s as Dynamic Helical Metallopolymers.

The study of metallopolymers with controllable helical sense remains in its infancy. We report arabinose-functionalized (Zn-salphen)-based conjugated polymers that display mirror-image circular dichroism spectra for L- and D-sugar sidechains respectively, signifying ordered (helical) coiling of the polymer backbone with opposite screw-sense preferences. The observation of different spectroscopic behavior and Cotton effects for a variety of solvents (in a reversible manner) and temperatures, ascribed to changes in the extent of intrachain (Zn⋅⋅⋅O(salphen) and π-stacking) interactions between Zn-salphen moieties, thus indicate the flexible, responsive and dynamic nature of the folded helical conformation in these systems. An application study signifying that activity can be governed by the structure and helical sense of the polymer is described.

Risk Assessment for Highly Pathogenic Avian Influenza A(H5N6/H5N8) Clade 2.3.4.4 Viruses.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Introduction of ORF3a-Q57H SARS-CoV-2 Variant Causing Fourth Epidemic Wave of COVID-19, Hong Kong, China.

We describe an introduction of clade GH severe acute respiratory syndrome coronavirus 2 causing a fourth wave of coronavirus disease in Hong Kong. The virus has an ORF3a-Q57H mutation, causing truncation of ORF3b. This virus evades induction of cytokine, chemokine, and interferon-stimulated gene expression in primary human respiratory cells.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface.

Attitudes Toward Retirement Drive the Effects of Retirement Preparation on Psychological and Physical Well-Being of Hong Kong Chinese Retirees Over Time.

Recent findings on retirement preparation found a positive impact on the psychological and physical well-being of retirees. However, the types of mental resources that are driving the relationship, such as attitudes toward retirement, only received limited attention. Reasoning from previous findings, we posit that attitudes toward retirement would explain the relationship between retirement preparation and well-being over time after retirement. A three-wave study was conducted in a sample of 130 Hong Kong Chinese retirees over a period of 1.5 years. Data were collected 6 months prior to retirement (T1) and 6 and 12 months after retirement (T2 and T3, respectively), in which preretirement preparation, attitudes toward retirement, and psychological and physical well-being were measured. The positive effect of T1 retirement planning on T3 physical and psychological well-being was partially mediated by T2 attitudes toward retirement. These results remain significant even after controlling for gender, education level, preretirement occupation, and well-being at T1. These findings reveal the role of attitudes toward retirement in driving postretirement adjustment over time.

Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses.

BACKGROUND: Neutrophil is of the most abundant number in human immune system. During acute influenza virus infection, neutrophils are already active in the early phase of inflammation - a time in which clinical biopsy or autopsy material is not readily available. However, the role of neutrophil in virus infection is not well understood. Here, we studied the role of neutrophil in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease. METHODS: Neutrophils were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro. The ability of the naïve neutrophils to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of neutrophils upon influenza virus infection were evaluated. RESULTS: Our results demonstrated that influenza virus infected alveolar epithelium allowed neutrophil transmigration. Significantly more neutrophils migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Neutrophils were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected neutrophils. H5N1 induced higher cytokine and chemokine gene transcription than H1N1 infected neutrophils, including TNF-α, IFN-ß, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was only observed in H1N1 infected neutrophils at 6 hpi while no NET formation was observed upon H5N1 infection. CONCLUSION: Our data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection and might contribute to the severity of H5N1 disease.

Therapeutic Implications of Human Umbilical Cord Mesenchymal Stromal Cells in Attenuating Influenza A(H5N1) Virus-Associated Acute Lung Injury.

Background: Highly pathogenic avian influenza viruses can cause severe forms of acute lung injury (ALI) in humans, where pulmonary flooding leads to respiratory failure. The therapeutic benefits of bone marrow mesenchymal stromal cells (MSCs) have been demonstrated in a model of ALI due to influenza A(H5N1) virus. However, clinical translation is impractical and limited by a decline in efficacy as the age of the donor increases. Umbilical cord MSCs (UC-MSCs) are easier to obtain by comparison, and their primitive source may offer more-potent therapeutic effects. Methods: Here we investigate the therapeutic efficacy of UC-MSCs on the mechanisms of pulmonary edema formation and alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs were also tested in a mouse model of influenza ALI. Results: We found that UC-MSCs were effective in restoring impaired alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs consistently outperformed bone marrow MSCs, partly because of greater growth factor secretion of angiopoietin 1 and hepatocyte growth factor. Conditioned UC-MSC medium and UC-MSC exosomes were also able to recapitulate these effects. However, UC-MSCs only slightly improved survival of A(H5N1)-infected mice. Conclusions: Our results suggest that UC-MSCs are effective in restoring alveolar fluid clearance and protein permeability in A(H5N1)-associated ALI and confer functional in addition to practical advantages over conventional bone marrow MSCs.

Risk Assessment of the Tropism and Pathogenesis of the Highly Pathogenic Avian Influenza A/H7N9 Virus Using Ex Vivo and In Vitro Cultures of Human Respiratory Tract.

BACKGROUND: Highly pathogenic avian influenza (HPAI)-H7N9 virus arising from low pathogenic avian influenza (LPAI)-H7N9 virus with polybasic amino acid substitutions in the hemagglutinin was detected in 2017. METHODS: We compared the tropism, replication competence, and cytokine induction of HPAI-H7N9, LPAI-H7N9, and HPAI-H5N1 in ex vivo human respiratory tract explants, in vitro culture of human alveolar epithelial cells (AECs) and pulmonary microvascular endothelial cells (HMVEC-L). RESULTS: Replication competence of HPAI- and LPAI-H7N9 were comparable in ex vivo cultures of bronchus and lung. HPAI-H7N9 predominantly infected AECs, whereas limited infection was observed in bronchus. The reduced tropism of HPAI-H7N9 in bronchial epithelium may explain the lack of human-to-human transmission despite a number of mammalian adaptation markers. Apical and basolateral release of virus was observed only in HPAI-H7N9- and H5N1-infected AECs regardless of infection route. HPAI-H7N9, but not LPAI-H7N9 efficiently replicated in HMVEC-L. CONCLUSIONS: Our findings demonstrate that a HPAI-H7N9 virus efficiently replicating in ex vivo cultures of human bronchus and lung. The HPAI-H7N9 was more efficient at replicating in human AECs and HMVEC-L than LPAI-H7N9 implying that endothelial tropism may involve in pathogenesis of HPAI-H7N9 disease.

Tropism of influenza B viruses in human respiratory tract explants and airway organoids.

Despite causing regular seasonal epidemics with substantial morbidity, mortality and socioeconomic burden, there is still a lack of research into influenza B viruses (IBVs). In this study, we provide for the first time a systematic investigation on the tropism, replication kinetics and pathogenesis of IBVs in the human respiratory tract.Physiologically relevant ex vivo explant cultures of human bronchus and lung, human airway organoids, and in vitro cultures of differentiated primary human bronchial epithelial cells and type-I-like alveolar epithelial cells were used to study the cellular and tissue tropism, replication competence and induced innate immune response of 16 IBV strains isolated from 1940 to 2012 in comparison with human seasonal influenza A viruses (IAVs), H1N1 and H3N2. IBVs from the diverged Yamagata- and Victoria-like lineages and the earlier undiverged period were included.The majority of IBVs replicated productively in human bronchus and lung with similar competence to seasonal IAVs. IBVs infected a variety of cell types, including ciliated cells, club cells, goblet cells and basal cells, in human airway organoids. Like seasonal IAVs, IBVs are low inducers of pro-inflammatory cytokines and chemokines. Most results suggested a higher preference for the conducting airway than the lower lung and strain-specific rather than lineage-specific pathogenicity of IBVs.Our results highlighted the non-negligible virulence of IBVs which require more attention and further investigation to alleviate the disease burden, especially when treatment options are limited.

Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Tropism and innate host responses of influenza A/H5N6 virus: an analysis of ex vivo and in vitro cultures of the human respiratory tract.

Since their first isolation in 2013, influenza A/H5N6 viruses have spread amongst poultry across multiple provinces in China and to Laos, Vietnam and Myanmar. So far, there have been 14 human H5N6 infections with 10 fatalities.We investigated the tropism, replication competence and cytokine induction of one human and two avian H5N6 isolates in ex vivo and in vitro cultures derived from the human respiratory tract. Virus tropism and replication were studied in ex vivo cultures of human nasopharynx, bronchus and lung. Induction of cytokines and chemokines was measured in vitro in virus-infected primary human alveolar epithelial cells.Human H5N6 virus replicated more efficiently than highly pathogenic avian influenza (HPAI) H5N1 virus and as efficiently as H1N1pdm in ex vivo human bronchus and lung and was also able to replicate in ex vivo cultures of human nasopharynx. Avian H5N6 viruses replicated less efficiently than H1N1pdm in human bronchial tissues and to similar titres as HPAI H5N1 in the lung. While the human H5N6 virus had affinity for avian-like receptors, the two avian isolates had binding affinity for both avian- and human-like receptors. All three H5N6 viruses were less potent inducers of pro-inflammatory cytokines compared with H5N1 virus.Human H5N6 virus appears better adapted to infect the human airways than H5N1 virus and may pose a significant public health threat.

Chelating σ-Aryl Post-Metallocenes: Probing Intramolecular [C-H···F-C] Interactions and Unusual Reaction Pathways.

Our interest in chelating σ-aryl ancillary ligands was motivated by their potential to impart unusual reactivity, since we envisioned that σ-donors with minimal π-donation would create a catalytic center with enhanced electrophilicity. We developed a family of Group 4 post-metallocene catalysts supported by pyridine-2-phenolate-6-(σ-aryl) [O,N,C] ligands bearing a fluorinated moiety in the vicinity of the metal. Notable features of these meta-substituted tris(hetero)aryl frameworks include their coordination geometry and inherent rigidity. For the first time, the elusive C-H···F-C interaction was manifested as NMR-discernible (1)H-(19)F coupling in solution and characterized by a neutron diffraction study. Their existence carries implications for catalyst design and in the context of weak attractive ligand-polymer interactions (WALPI), since they substantiate the practical viability of the ortho-F···H(ß) ligand-polymer interactions proposed for living Group 4 fluorinated bis(phenoxyimine) catalysts. In metal-catalyzed olefin polymerization reactions, the notion of noncovalent interactions between an active ancillary ligand and the growing polymer chain is new. These interactions must be fragile and transient in nature, otherwise the intrinsic chain propagation process would be disrupted, and inherently tunable attractive forces such as hydrogen bonds are ideally suited to this role. The nature, relevance, and usability of extremely weak hydrogen bonds such as C-H···F-C has been a topical yet controversial area of research. We subsequently prepared a series of Group 4 complexes supported by fluorinated (σ-aryl)-2-phenolate-6-pyridyl [O,C,N] ligands. [(1)H,(19)F]-HMBC NMR experiments were conducted to probe the observed (1)H-(19)F coupling, and specifically separate contributions from scalar (J) coupling and cross-correlation (CR) interference. For the first time, a significant scalar component was confirmed for the (1)H-(19)F coupling in Ti-[O,C,N] and [O,N,C] complexes, which occurs with chemical connectivity across intramolecular C-H···F-C interactions. This result is important because the applicability of weak attractive ligand-polymer interactions in catalysis is feasible only if the observed coupling and hence the noncovalent interaction is genuine. The verified intramolecular C-H···F-C contacts in these complexes can therefore be considered as synthetic models for ligand-polymer interactions in olefin polymerization processes. Significantly, reports concerning late transition metal systems have appeared that hint at the generality of the WALPI concept for modulating polymerization reactions. We evaluated the olefin polymerization reactivity of Ti-[O,N,C] catalysts through judicious substitution. DFT calculations, which revealed diverse kinetically competitive reaction pathways and active sites (including unusual ethylene-assimilated species) in addition to normal chain propagation, were also employed to rationalize polymerization efficiencies. Further developments in catalytic applications of multidentate σ-aryl ligand systems and novel reactivity of the corresponding complexes can be envisaged.