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.

Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.

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.

Reduced Pathogenicity and Transmission Potential of Omicron BA.1 and BA.2 Sublineages Compared with the Early Severe Acute Respiratory Syndrome Coronavirus 2 D614G Variant in Syrian Hamsters.

BACKGROUND: The epidemiological advantage of Omicron variant is evidenced by its rapid spread and the ability to outcompete prior variants. Among Omicron sublineages, early outbreaks were dominated by BA.1, while BA.2 has gained dominance since February 2022. The relative pathogenicity and transmissibility of BA.1 and BA.2 have not been fully defined. METHODS: We compared viral loads and clinical signs in Syrian hamsters after infection with BA.1, BA.2, or D614G variant. A competitive transmission model and next-generation sequencing were used to compare the relative transmission potential of BA.1 and BA.2. RESULTS: BA.1 and BA.2 caused no apparent clinical signs, while D614G caused more than 10% weight loss. Higher viral loads were detected in nasal wash samples and nasal turbinate and lung tissues from BA.1-inoculated hamsters compared with BA.2-inoculated hamsters. No aerosol transmission was observed for BA.1 or BA.2 under the experimental condition in which D614G transmitted efficiently. BA.1 and BA.2 were able to transmit among hamsters via direct contact; however, BA.1 transmitted more efficiently than BA.2 under the competitive transmission model. No recombination was detected from direct contacts exposed simultaneously to BA.1 and BA.2. CONCLUSIONS: Omicron BA.1 and BA.2 demonstrated attenuated pathogenicity and reduced transmission potential in hamsters compared with early SARS-CoV-2 strains.

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.

SARS-CoV-2 infection aggravates cigarette smoke-exposed cell damage in primary human airway epithelia.

BACKGROUND: The coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic with over 627 million cases and over 6.5 million deaths. It was reported that smoking-related chronic obstructive pulmonary disease (COPD) might be a crucial risk for COVID-19 patients to develop severe condition. As cigarette smoke (CS) is the major risk factor for COPD, we hypothesize that barrier dysfunction and an altered cytokine response in CS-exposed airway epithelial cells may contribute to increased SARS-CoV-2-induced immune response that may result in increased susceptibility to severe disease. The aim of this study was to evaluate the role of CS on SARS-CoV-2-induced immune and inflammatory responses, and epithelial barrier integrity leading to airway epithelial damage. METHODS: Primary human airway epithelial cells were differentiated under air-liquid interface culture. Cells were then exposed to cigarette smoke medium (CSM) before infection with SARS-CoV-2 isolated from a local patient. The infection susceptibility, morphology, and the expression of genes related to host immune response, airway inflammation and damages were evaluated. RESULTS: Cells pre-treated with CSM significantly caused higher replication of SARS-CoV-2 and more severe SARS-CoV-2-induced cellular morphological alteration. CSM exposure caused significant upregulation of long form angiotensin converting enzyme (ACE)2, a functional receptor for SARS-CoV-2 viral entry, transmembrane serine protease (TMPRSS)2 and TMPRSS4, which cleave the spike protein of SARS-CoV-2 to allow viral entry, leading to an aggravated immune response via inhibition of type I interferon pathway. In addition, CSM worsened SARS-CoV-2-induced airway epithelial cell damage, resulting in severe motile ciliary disorder, junctional disruption and mucus hypersecretion. CONCLUSION: Smoking led to dysregulation of host immune response and cell damage as seen in SARS-CoV-2-infected primary human airway epithelia. These findings may contribute to increased disease susceptibility with severe condition and provide a better understanding of the pathogenesis of SARS-CoV-2 infection in smokers.

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury.

Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.

Role of Epithelial-Endothelial Cell Interaction in the Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health globally. Patients with severe COVID-19 disease progress to acute respiratory distress syndrome, with respiratory and multiple organ failure. It is believed that dysregulated production of proinflammatory cytokines and endothelial dysfunction contribute to the pathogenesis of severe diseases. However, the mechanisms of SARS-CoV-2 pathogenesis and the role of endothelial cells are poorly understood. METHODS: Well-differentiated human airway epithelial cells were used to explore cytokine and chemokine production after SARS-CoV-2 infection. We measured the susceptibility to infection, immune response, and expression of adhesion molecules in human pulmonary microvascular endothelial cells (HPMVECs) exposed to conditioned medium from infected epithelial cells. The effect of imatinib on HPMVECs exposed to conditioned medium was evaluated. RESULTS: We demonstrated the production of interleukin-6, interferon gamma-induced protein-10, and monocyte chemoattractant protein-1 from the infected human airway cells after infection with SARS-CoV-2. Although HPMVECs did not support productive replication of SARS-CoV-2, treatment of HPMVECs with conditioned medium collected from infected airway cells induced an upregulation of proinflammatory cytokines, chemokines, and vascular adhesion molecules. Imatinib inhibited the upregulation of these cytokines, chemokines, and adhesion molecules in HPMVECs treated with conditioned medium. CONCLUSIONS: We evaluated the role of endothelial cells in the development of clinical disease caused by SARS-CoV-2 and the importance of endothelial cell-epithelial cell interaction in the pathogenesis of human COVID-19 diseases.

Cellular tropism of SARS-CoV-2 in the respiratory tract of Syrian hamsters and B6.Cg-Tg(K18-ACE2)2Prlmn/J transgenic mice.

Several animal models have been developed to study the pathophysiology of SARS-CoV-2 infection and to evaluate vaccines and therapeutic agents for this emerging disease. Similar to infection with SARS-CoV-1, infection of Syrian hamsters with SARS-CoV-2 results in moderate respiratory disease involving the airways and lung parenchyma but does not lead to increased mortality. Using a combination of immunohistochemistry and transmission electron microscopy, we showed that the epithelium of the conducting airways of hamsters was the primary target for viral infection within the first 5 days of infection, with little evidence of productive infection of pneumocytes. At 6 days postinfection, antigen was cleared but parenchymal damage persisted, and the major pathological changes resolved by day 14. These findings are similar to those previously reported for hamsters with SARS-CoV-1 infection. In contrast, infection of K18-hACE2 transgenic mice resulted in pneumocyte damage, with viral particles and replication complexes in both type I and type II pneumocytes together with the presence of convoluted or cubic membranes; however, there was no evidence of virus replication in the conducting airways. The Syrian hamster is a useful model for the study of SARS-CoV-2 transmission and vaccination strategies, whereas infection of the K18-hCE2 transgenic mouse results in lethal disease with fatal neuroinvasion but with sparing of conducting airways.

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.

Postmortem Stability of SARS-CoV-2 in Mouse Lung Tissue.

The infectivity of severe acute respiratory syndrome coronavirus 2 in deceased persons and organisms remains unclear. We studied transgenic K18 hACE2 mice to determine the kinetics of virus infectivity after host death. Five days after death, virus infectivity in the lung declined by >96% and RNA copies declined by 48.2%.

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.

Pathophysiology of infection with SARS-CoV-2-What is known and what remains a mystery.

Coronavirus disease 2019 (COVID-19), caused by coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused extensive disruption and mortality since its recent emergence. Concomitantly, there has been a race to understand the virus and its pathophysiology. The clinical manifestations of COVID-19 are manifold and not restricted to the respiratory tract. Extrapulmonary manifestations involving the gastrointestinal tract, hepatobiliary system, cardiovascular and renal systems have been widely reported. However, the pathophysiology of many of these manifestations is controversial with questionable support for direct viral invasion and an abundance of alternative explanations such as pre-existing medical conditions and critical illness. Prior research on SARS-Co-V and NL63 was rapidly leveraged to identify angiotensin-converting enzyme 2 (ACE2) receptor as the key cell surface receptor for SARS-CoV-2. The distribution of ACE2 has been used as a starting point for estimating vulnerability of various tissue types to SARS-CoV-2 infection. Sophisticated organoid and animal models have been used to demonstrate such infectivity of extrapulmonary tissues in vitro, but the clinical relevance of these findings remains uncertain. Clinical autopsy studies are typically small and inevitably biased towards patients with severe COVID-19 and prolonged hospitalization. Technical issues such as delay between time of death and autopsy, use of inappropriate antibodies for paraffin-embedded tissue sections and misinterpretation of cellular structures as virus particles on electron micrograph images are additional problems encountered in the extant literature. Given that SARS-CoV-2 is likely to circulate permanently in human populations, there is no doubt that further work is required to clarify the pathobiology of COVID-19.

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.

Terahertz optical thickness and birefringence measurement for thermal barrier coating defect location.

We present a normal incidence terahertz reflectivity technique to determine the optical thickness and birefringence of yttria-stabilized zirconia (YSZ) thermal barrier coatings (TBCs). Initial verification of the method was achieved by measurement of a set of fused silica calibration samples with known thicknesses and showed excellent agreement (<1% of refractive index) with the literature. The THz-measured optical thickness and its variation through the depth profile of the YSZ coating are shown to be in good agreement (<4%) with scanning electron microscope cross-sectional thickness measurements. In addition, the position of discontinuities in both the optical thickness and birefringence appear to be correlated to coating failure points observed during accelerated aging trials.

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.