Divergent trajectory of replication and intrinsic pathogenicity of SARS-CoV-2 Omicron post-BA.2/5 subvariants in the upper and lower respiratory tract.

BACKGROUND: Earlier Omicron subvariants including BA.1, BA.2, and BA.5 emerged in waves, with a subvariant replacing the previous one every few months. More recently, the post-BA.2/5 subvariants have acquired convergent substitutions in spike that facilitated their escape from humoral immunity and gained ACE2 binding capacity. However, the intrinsic pathogenicity and replication fitness of the evaluated post-BA.2/5 subvariants are not fully understood. METHODS: We systemically investigated the replication fitness and intrinsic pathogenicity of representative post-BA.2/5 subvariants (BL.1, BQ.1, BQ.1.1, XBB.1, CH.1.1, and XBB.1.5) in weanling (3-4 weeks), adult (8-10 weeks), and aged (10-12 months) mice. In addition, to better model Omicron replication in the human nasal epithelium, we further investigated the replication capacity of the post-BA.2/5 subvariants in human primary nasal epithelial cells. FINDINGS: We found that the evaluated post-BA.2/5 subvariants are consistently attenuated in mouse lungs but not in nasal turbinates when compared with their ancestral subvariants BA.2/5. Further investigations in primary human nasal epithelial cells revealed a gained replication fitness of XBB.1 and XBB.1.5 when compared to BA.2 and BA.5.2. INTERPRETATION: Our study revealed that the post-BA.2/5 subvariants are attenuated in lungs while increased in replication fitness in the nasal epithelium, indicating rapid adaptation of the circulating Omicron subvariants in the human populations. FUNDING: The full list of funding can be found at the Acknowledgements section.

Correlations of Myeloperoxidase (MPO), Adenosine deaminase (ADA), C-C motif chemokine 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in the nasopharyngeal specimens with the diagnosis and severity of SARS-CoV-2 infections.

Cytokine dynamics in patients with coronavirus disease 2019 (COVID-19) have been studied in blood but seldomly in respiratory specimens. We studied different cell markers and cytokines in fresh nasopharyngeal swab specimens for the diagnosis and for stratifying the severity of COVID-19. This was a retrospective case-control study comparing Myeloperoxidase (MPO), Adenosine deaminase (ADA), C-C motif chemokine ligand 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in 490 (327 patients and 163 control) nasopharyngeal specimens from 317 (154 COVID-19 and 163 control) hospitalized patients. Of the 154 COVID-19 cases, 46 died. Both total and normalized MPO, ADA, CCL22, TNFα, and IL-6 mRNA expression levels were significantly higher in the nasopharyngeal specimens of infected patients when compared with controls, with ADA showing better performance (OR 5.703, 95% CI 3.424-9.500, p < 0.001). Receiver operating characteristics (ROC) curve showed that the cut-off value of normalized ADA mRNA level at 2.37 × 10-3 had a sensitivity of 81.8% and specificity of 83.4%. While patients with severe COVID-19 had more respiratory symptoms, and elevated lactate dehydrogenase, multivariate analysis showed that severe COVID-19 patients had lower CCL22 mRNA (OR 0.211, 95% CI 0.060-0.746, p = 0.016) in nasopharyngeal specimens, while lymphocyte count, C-reactive protein, and viral load in nasopharyngeal specimens did not correlate with disease severity. In summary, ADA appears to be a better biomarker to differentiate between infected and uninfected patients, while CCL22 has the potential in stratifying the severity of COVID-19.

Air dispersal of respiratory viruses other than severe acute respiratory coronavirus virus 2 (SARS-CoV-2) and the implication on hospital infection control.

BACKGROUND: Air dispersal of respiratory viruses other than SARS-CoV-2 has not been systematically reported. The incidence and factors associated with air dispersal of respiratory viruses are largely unknown. METHODS: We performed air sampling by collecting 72,000 L of air over 6 hours for pediatric and adolescent patients infected with parainfluenza virus 3 (PIF3), respiratory syncytial virus (RSV), rhinovirus, and adenovirus. The patients were singly or 2-patient cohort isolated in airborne infection isolation rooms (AIIRs) from December 3, 2021, to January 26, 2022. The viral load in nasopharyngeal aspirates (NPA) and air samples were measured. Factors associated with air dispersal were investigated and analyzed. RESULTS: Of 20 singly isolated patients with median age of 30 months (range, 3 months-15 years), 7 (35%) had air dispersal of the viruses compatible with their NPA results. These included 4 (40%) of 10 PIF3-infected patients, 2 (66%) of 3 RSV-infected patients, and 1 (50%) of 2 adenovirus-infected patients. The mean viral load in their room air sample was 1.58×103 copies/mL. Compared with 13 patients (65%) without air dispersal, these 7 patients had a significantly higher mean viral load in their NPA specimens (6.15×107 copies/mL vs 1.61×105 copies/mL; P < .001). Another 14 patients were placed in cohorts as 7 pairs infected with the same virus (PIF3, 2 pairs; RSV, 3 pairs; rhinovirus, 1 pair; and adenovirus, 1 pair) in double-bed AIIRs, all of which had air dispersal. The mean room air viral load in 2-patient cohorts was significantly higher than in rooms of singly isolated patients (1.02×104 copies/mL vs 1.58×103 copies/mL; P = .020). CONCLUSION: Air dispersal of common respiratory viruses may have infection prevention and public health implications.

Emerging Infections Due to Shewanella spp.: A Case Series of 128 Cases Over 10 Years.

Background: Shewanella species are emerging pathogens that can cause severe hepatobiliary, skin and soft tissue, gastrointestinal, respiratory infections, and bacteremia. Here we reported the largest case series of infections caused by Shewanella species. Aim: To identify the clinical features and risk factors predisposing to Shewanella infections. To evaluate resistance pattern of Shewanella species and appropriateness of antibiotic use in the study cohort. Methods: Patients admitted to a regional hospital in Hong Kong with Shewanella species infection from April 1, 2010 to December 31, 2020 were included. Demographics, antibiotics, microbiology, and outcomes were retrospectively analyzed. Findings: Over the 10 years, we identified 128 patients with Shewanella species infection. 61.7% were male with a median age of 78 (IQR 65-87). Important underlying diseases included hepatobiliary diseases (63.3%), malignancy (26.6%), chronic kidney disease or end-stage renal failure (25.8%), and diabetes mellitus (22.7%). Hepatobiliary infections (60.4%) were the most common clinical manifestation. Majority (92.2%) were infected with Shewanella algae, while 7.8% were infected with Shewanella putrefaciens. The identified organisms were usually susceptible to ceftazidime (98.7%), gentamicin (97.4%), cefoperazone-sulbactam (93.5%) and ciprofloxacin (90.3%). Imipenem-susceptible strains were only present in 76.6% of isolates. Conclusion: This largest case series suggested that Shewanella infections are commonly associated with underlying comorbidities, especially with hepatobiliary diseases and malignancy. Although Shewanella species remained largely susceptible to third and fourth generation cephalosporins and aminoglycosides, carbapenem resistance has been on a significant rise.

Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals.

Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA, adenoviral vector and inactivated vaccines fail to induce. Here, we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene, which encodes 2'-O-methyltransferase, is catalytically disrupted by a point mutation. This virus, designated d16, was severely attenuated in hamsters and transgenic mice, causing only asymptomatic and nonpathogenic infection. A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters, thus preventing viral spread in a contact-based transmission model. It also robustly stimulated humoral and cell-mediated immune responses, thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model. The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants. Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice. Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain, to which new features might be introduced to improve safety, transmissibility, immunogenicity and efficacy.

Temporal trends and patterns of infective endocarditis in a Chinese population: A territory-wide study in Hong Kong (2002-2019).

Background: The characteristics of infective endocarditis (IE) in Asians are poorly understood. Therefore, we aim to describe the epidemiological trends and clinical features of IE in Hong Kong. Methods: All patients with incident IE from 2002-2019 in a territory-wide clinical database in Hong Kong were identified. We studied the age- and sex-adjusted and one-year mortality of IE between 2002 and 2019 and identified significant contributors to 1-year all-cause death using the attributable fraction. We used propensity score and inverse propensity of treatment weighting to study the association of surgery with mortality. Findings: A total of 5139 patients (60.4 ± 18.2years, 37% women) were included. The overall incidence of IE was 4.9 per 100,000 person-year, which did not change over time (P = 0.17). Patients in 2019 were older and more comorbid than those in 2002. The one-year crude mortality rate was 30% in 2002, which did not change significantly over time (P = 0.10). Between 2002 and 2019, the rate of surgery increased and was associated with a 51% risk reduction in 1-year all-cause mortality (Hazard Ratio 0.49 [0.28-0.87], P = 0.015). Advanced age (attributable fraction 19%) and comorbidities (attributable fraction 15%) were significant contributors to death. Interpretation: The incidence of IE in Hong Kong did not change between 2002 and 2019. Patients with IE in 2019 were older and had more comorbidities than those in 2002. Mortality of IE remains persistently high in Hong Kong. Together, these data can guide public health strategies to improve the outcomes of patients with IE. Funding: This work was supported by Sanming Project of Medicine in Shenzhen, China [No. SZSM201911020] and HKU-SZH Fund for Shenzhen Key Medical Discipline [No. SZXK2020081].

Fusion-inhibition peptide broadly inhibits influenza virus and SARS-CoV-2, including Delta and Omicron variants.

Pandemic influenza virus and SARS-CoV-2 vaiants have posed major global threats to public health. Broad-spectrum antivirals blocking viral entry can be an effective strategy for combating these viruses. Here, we demonstrate a frog-defensin-derived basic peptide (FBP), which broadly inhibits the influenza virus by binding to haemagglutinin so as to block low pH-induced HA-mediated fusion and antagonizes endosomal acidification to inhibit the influenza virus. Moreover, FBP can bind to the SARS-CoV-2 spike to block spike-mediated cell-cell fusion in 293T/ACE2 cells endocytosis. Omicron spike shows a weak cell-cell fusion mediated by TMPRSS2 in Calu3 cells, making the Omicron variant sensitive to endosomal inhibitors. In vivo studies show that FBP broadly inhibits the A(H1N1)pdm09 virus in mice and SARS-CoV-2 (HKU001a and Delta)in hamsters. Notably, FBP shows significant inhibition of Omicron variant replication even though it has a high number of mutations in spike. In conclusion, these results suggest that virus-targeting FBP with a high barrier to drug resistance can be an effective entry-fusion inhibitor against influenza virus and SARS-CoV-2 in vivo.

Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model.

BACKGROUND: Post-vaccination myopericarditis is reported after immunization with coronavirus disease 2019 (COVID-19) messenger RNA (mRNA) vaccines. The effect of inadvertent intravenous injection of this vaccine on the heart is unknown. METHODS: We compared the clinical manifestations, histopathological changes, tissue mRNA expression, and serum levels of cytokine/chemokine and troponin in Balb/c mice at different time points after intravenous (IV) or intramuscular (IM) vaccine injection with normal saline (NS) control. RESULTS: Although significant weight loss and higher serum cytokine/chemokine levels were found in IM group at 1-2 days post-injection (dpi), only IV group developed histopathological changes of myopericarditis as evidenced by cardiomyocyte degeneration, apoptosis, and necrosis with adjacent inflammatory cell infiltration and calcific deposits on visceral pericardium, although evidence of coronary artery or other cardiac pathologies was absent. Serum troponin level was significantly higher in IV group. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen expression by immunostaining was occasionally found in infiltrating immune cells of the heart or injection site, in cardiomyocytes and intracardiac vascular endothelial cells, but not skeletal myocytes. The histological changes of myopericarditis after the first IV-priming dose persisted for 2 weeks and were markedly aggravated by a second IM- or IV-booster dose. Cardiac tissue mRNA expression of interleukin (IL)-1ß, interferon (IFN)-ß, IL-6, and tumor necrosis factor (TNF)-α increased significantly from 1 dpi to 2 dpi in the IV group but not the IM group, compatible with presence of myopericarditis in the IV group. Ballooning degeneration of hepatocytes was consistently found in the IV group. All other organs appeared normal. CONCLUSIONS: This study provided in vivo evidence that inadvertent intravenous injection of COVID-19 mRNA vaccines may induce myopericarditis. Brief withdrawal of syringe plunger to exclude blood aspiration may be one possible way to reduce such risk.

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells.

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

Insights from a Prospective Follow-up of Thyroid Function and Autoimmunity among COVID-19 Survivors.

BACKGROUND: The occurrence of Graves' disease and Hashimoto thyroiditis after coronavirus disease 2019 (COVID-19) raised concerns that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may trigger thyroid autoimmunity. We aimed to address the current uncertainties regarding incident thyroid dysfunction and autoimmunity among COVID-19 survivors. METHODS: We included consecutive adult COVID-19 patients without known thyroid disorders, who were admitted to Queen Mary Hospital from July 21 to September 21, 2020 and had serum levels of thyroid-stimulating hormone, free thyroxine, free triiodothyronine (fT3), and anti-thyroid antibodies measured both on admission and at 3 months. RESULTS: In total, 122 patients were included. Among 20 patients with abnormal thyroid function tests (TFTs) on admission (mostly low fT3), 15 recovered. Among 102 patients with initial normal TFTs, two had new-onset abnormalities that could represent different phases of thyroiditis. Among 104 patients whose anti-thyroid antibody titers were reassessed, we observed increases in anti-thyroid peroxidase (TPO) (P<0.001) and anti-thyroglobulin (P<0.001), but not anti-thyroid stimulating hormone receptor titers (P=0.486). Of 82 patients with negative anti-TPO findings at baseline, 16 had a significant interval increase in anti-TPO titer by >12 U, and four became anti-TPO-positive. Worse baseline clinical severity (P=0.018), elevated C-reactive protein during hospitalization (P=0.033), and higher baseline anti-TPO titer (P=0.005) were associated with a significant increase in anti-TPO titer. CONCLUSION: Most patients with thyroid dysfunction on admission recovered during convalescence. Abnormal TFTs suggestive of thyroiditis occurred during convalescence, but infrequently. Importantly, our novel observation of an increase in anti-thyroid antibody titers post-COVID-19 warrants further follow-up for incident thyroid dysfunction among COVID-19 survivors.

Differential role of sphingomyelin in influenza virus, rhinovirus and SARS-CoV-2 infection of Calu-3 cells.

Host cell lipids play a pivotal role in the pathogenesis of respiratory virus infection. However, a direct comparison of the lipidomic profile of influenza virus and rhinovirus infections is lacking. In this study, we first compared the lipid profile of influenza virus and rhinovirus infection in a bronchial epithelial cell line. Most lipid features were downregulated for both influenza virus and rhinovirus, especially for the sphingomyelin features. Pathway analysis showed that sphingolipid metabolism was the most perturbed pathway. Functional study showed that bacterial sphingomyelinase suppressed influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, but promoted rhinovirus replication. These findings suggest that sphingomyelin pathway can be a potential target for antiviral therapy, but should be carefully evaluated as it has opposite effects on different respiratory viruses. Furthermore, the differential effect of sphingomyelinase on rhinovirus and influenza virus may explain the interference between rhinovirus and influenza virus infection.

Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein.

SARS-CoV-2 is of zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction of the spike protein. Here, we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in Vero E6 cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no obvious pathological changes and does not induce elevation of proinflammatory cytokines, but still triggers a strong neutralizing antibody and T cell response in hamsters and mice. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, with little sign of virus replication in the upper or lower respiratory tract, demonstrating sterilizing immunity.

Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR.

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFY™) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs. 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Host and viral determinants for efficient SARS-CoV-2 infection of the human lung.

Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we analyze host and viral determinants essential for efficient SARS-CoV-2 infection in both human lung epithelial cells and ex vivo human lung tissues. We identify heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Next, we show that sialic acids present on ACE2 prevent efficient spike/ACE2-interaction. While SARS-CoV infection is substantially limited by the sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissues, infection by SARS-CoV-2 is limited to a lesser extent. We further demonstrate that the furin-like cleavage site in SARS-CoV-2 spike is required for efficient virus replication in human lung but not intestinal tissues. These findings provide insights on the efficient SARS-CoV-2 infection of human lungs.

A child with acute myeloid leukemia complicated by calcaneal osteomyelitis due to Mycobacterium abscessus infection after induction chemotherapy successfully salvaged with bedaquiline and clofazimine.

Our patient was a 4-year-old female with acute myeloid leukemia complicated with right calcaneal osteomyelitis due to Mycobacterium abscessus with subcutaneous abscesses extending to the popliteal and groin regions after two courses of induction chemotherapy according to NOPHO-AML 2012 protocol. She required multiple operations and prolonged anti-mycobacterial therapy. A high index of suspicion for mycobacterial infection is required for immunocompromised patients with prolonged fever or unusual presentation. Mycobacterial osteomyelitis is rare, difficult to diagnose and treat, and may necessitate prolonged interruption of anti-leukemic therapy. Multidisciplinary collaboration in patient management is crucial. Long-term toxicity of antimicrobials with uncertain efficacy should not be overlooked.

Thyroid Dysfunction in Relation to Immune Profile, Disease Status, and Outcome in 191 Patients with COVID-19.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related thyroiditis is increasingly recognized. The role of thyroid autoimmunity and SARS-CoV-2 viral load in SARS-CoV-2-related thyroid dysfunction is unclear. We evaluated the thyroid function of a cohort of coronavirus disease 2019 (COVID-19) patients, in relation to their clinical features, and biochemical, immunological, and inflammatory markers. METHODS: Consecutive adult patients, without known thyroid disorders, admitted to Queen Mary Hospital for COVID-19 from July 21 to August 21, 2020, were included. Serum levels of thyroid-stimulating hormone (TSH), free thyroxine, free triiodothyronine (fT3), and antithyroid antibodies were measured on admission. RESULTS: Among 191 patients with COVID-19 (mean age 53.5 ±â€…17.2 years; 51.8% male), 84.3% were mild, 12.6% were moderate, and 3.1% were severe. Abnormal thyroid function was seen in 13.1%. Ten patients had isolated low TSH, suggestive of subclinical thyrotoxicosis due to thyroiditis, although the contribution of autoimmunity was likely in 2 of them. Autoimmune thyroiditis probably also contributed to subclinical hypothyroidism in another patient. Ten patients had isolated low fT3, likely representing nonthyroidal illness syndrome. Lower SARS-Cov-2 polymerase chain reaction cycle threshold values and elevated C-reactive protein were independently associated with occurrence of low TSH (P = .030) and low fT3 (P = .007), respectively. A decreasing trend of fT3 with increasing COVID-19 severity (P = .032) was found. Patients with low fT3 had more adverse COVID-19-related outcomes. CONCLUSION: Around 15% of patients with mild to moderate COVID-19 had thyroid dysfunction. There may be a direct effect of SARS-CoV-2 on thyroid function, potentially leading to exacerbation of pre-existing autoimmune thyroid disease. Low fT3, associated with systemic inflammation, may have a prognostic significance.

Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target.

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

Olfactory Dysfunction in Coronavirus Disease 2019 Patients: Observational Cohort Study and Systematic Review.

BACKGROUND: Olfactory dysfunction (OD) has been reported in coronavirus disease 2019 (COVID-19). However, there are knowledge gaps about the severity, prevalence, etiology, and duration of OD in COVID-19 patients. METHODS: Olfactory function was assessed in all participants using questionnaires and the butanol threshold test (BTT). Patients with COVID-19 and abnormal olfaction were further evaluated using the smell identification test (SIT), sinus imaging, and nasoendoscopy. Selected patients received nasal biopsies. Systematic review was performed according to PRISMA guidelines. PubMed items from January 1, 2020 to April 23, 2020 were searched. Studies that reported clinical data on olfactory disturbances in COVID-19 patients were analyzed. RESULTS: We included 18 COVID-19 patients and 18 controls. Among COVID-19 patients, 12 of 18 (67%) reported olfactory symptoms and OD was confirmed in 6 patients by BTT and SIT. Olfactory dysfunction was the only symptom in 2 patients. Mean BTT score of patients was worse than controls (P = .004, difference in means = 1.8; 95% confidence interval, 0.6-2.9). Sinusitis and olfactory cleft obstruction were absent in most patients. Immunohistochemical analysis of nasal biopsy revealed the presence of infiltrative CD68+ macrophages harboring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen in the stroma. Olfactory dysfunction persisted in 2 patients despite clinical recovery. Systematic review showed that the prevalence of olfactory disturbances in COVID-19 ranged from 5% to 98%. Most studies did not assess olfaction quantitatively. CONCLUSIONS: Olfactory dysfunction is common in COVID-19 and may be the only symptom. Coronavirus disease 2019-related OD can be severe and prolonged. Mucosal infiltration by CD68+ macrophages expressing SARS-CoV-2 viral antigen may contribute to COVID-19-related OD.

Evaluating the use of posterior oropharyngeal saliva in a point-of-care assay for the detection of SARS-CoV-2.

During the Coronavirus disease 2019 (COVID-19) pandemic, logistic problems associated with specimen collection limited the SARS-CoV-2 testing, especially in the community. In this study, we assessed the use of posterior oropharyngeal saliva as specimens for the detection of SARS-CoV-2 in an automated point-of-care molecular assay. Archived nasopharyngeal swab (NPS) and posterior oropharyngeal saliva specimens of 58 COVID-19 patients were tested with the Xpert® Xpress SARS-CoV-2 assay. SARS-CoV-2 was detected in either NPS or saliva specimens of all patients. Among them, 84.5% (49/58) tested positive in both NPS and saliva, 10.3% (6/58) tested positive in NPS only, and 5.2% (3/58) tested positive in saliva only. No significant difference in the detection rate was observed between NPS and saliva (McNemar's test p = 0.5078). The detection rate was slightly higher for N2 (NPS 94.8% and Saliva 93.1%) than that of the E gene target (Saliva: 89.7% vs 82.8%) on both specimen types. Significantly earlier median Ct value was observed for NPS comparing to that of saliva on both E (26.8 vs 29.7, p = 0.0002) and N2 gene target (29.3 vs 32.3, p = 0.0002). The median Ct value of E gene target was significantly earlier than that of the N2 gene target for both NPS (26.8 vs 29.3, p < 0.0001) and saliva (29.7 vs 32.3, p < 0.0001). In conclusion, posterior oropharyngeal saliva and NPS were found to have similar detection rates in the point-of-care test for SARS-CoV-2 detection. Since posterior oropharyngeal saliva can be collected easily, the use of saliva as an alternative specimen type for SARS-CoV-2 detection is recommended.

Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages.

BACKGROUND: Human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) poses an ongoing threat to public health worldwide. The studies of MERS patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of MERS-CoV. We sought to identify pattern recognition receptor (PRR) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon MERS-CoV infection. METHODS: The potential signaling pathways were manipulated individually by pharmacological inhibition, small interfering ribonucleic acid (siRNA) depletion, and antibody blocking. The MERS-CoV-induced proinflammatory response was evaluated by measuring the expression levels of key cytokines and/or chemokines. Reverse transcription-quantitative polymerase chain reaction assay, flow cytometry analysis, and Western blotting were applied to evaluate the activation of related PRRs and engagement of adaptors. RESULTS: MERS-CoV replication significantly upregulated C-type lectin receptor (CLR) macrophage-inducible Ca2+-dependent lectin receptor (Mincle). The role of Mincle for MERS-CoV-triggered cytokine/chemokine induction was established based on the results of antibody blockage, siRNA depletion of Mincle and its adaptor spleen tyrosine kinase (Syk), and Syk pharmacological inhibition. The cytokine and/or chemokine induction was significantly attenuated by siRNA depletion of retinoic acid-inducible-I-like receptors (RLR) or adaptor, indicating that RLR signaling also contributed to MERS-CoV-induced proinflammatory response. CONCLUSIONS: The CLR and RLR pathways are activated and contribute to the proinflammatory response in MERS-CoV-infected macrophages.