Analysis of pathogen detection results of upper respiratory tract specimens from fever clinic patients

Objective: To explore the pathogen composition and distribution characteristics of pathogens in respiratory samples from patients with fever of unknown origin. Methods: A total of 96 respiratory samples of patients with unknown cause fever with respiratory symptoms were collected from four hospitals above grade II in Shijiazhuang area (Hebei Provincial Hospital of Traditional Chinese Medicine, Luancheng District People's Hospital, Luquan District People's Hospital, Shenze County Hospital) from January to April 2020, and multiplex-fluorescent polymerase chain reaction(PCR)was used to detect influenza A virus, influenza B virus, enterovirus, parainfluenza virus I/II/III/IV, respiratory adenovirus, human metapneumovirus, respiratory syncytial virus, human rhinovirus, human bocavirus, COVID-19, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, Group A streptococcus, Haemophilus influenzae, Staphylococcus aureus nucleic acid detection, the results were analyzed for chi-square. Results: A total of 8 pathogens were detected in the upper respiratory tract samples of 96 fever patients, including 1 kind of virus, 6 kinds of bacterias, and Mycoplasma pneumoniae. There were 12 viruses including influenza virus and parainfluenza virus, Legionella pneumophila and Chlamydia pneumoniae were not detected. The pathogen detection rates in descending order were Streptococcus pneumoniae (58/96, 60.42%), Haemophilus influenzae(38/96, 39.58%), Klebsiella pneumoniae (14/96, 14.58%), Staphylococcus aureus (10/96, 10.42%), Mycoplasma pneumoniae (8/96, 8.33%), Pseudomonas aeruginosa (6/96, 6.25%), Group A streptococcus (4/96, 4.17%) and human rhinovirus (2/96, 2.08%). The proportions of single-pathogen infection and multi-pathogen mixed infection in fever clinic patients were similar, 41.67% (40/96) and 45.83% (44/96), respectively, and 12.50% (12/96)of the cases had no pathogens detected. The infection rate of Mycoplasma pneumoniae in female patients with fever (21.43%) was higher than that in male patients with fever (2.94%) (P < 0.05). There was no statistical difference between the distribution of of other pathogens and gender and age(P > 0.05). Conclusions: The upper respiratory tract pathogens were mainly bacterial infections, and occasional human rhinovirus and Mycoplasma pneumonia infections. In clinical diagnosis and treatment, comprehensive consideration should be given to the pathogen detection.

Bioactive compounds from Huashi Baidu decoction possess both antiviral and anti-inflammatory effects against COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health concern, and effective antiviral reagents are urgently needed. Traditional Chinese medicine theory-driven natural drug research and development (TCMT-NDRD) is a feasible method to address this issue as the traditional Chinese medicine formulae have been shown effective in the treatment of COVID-19. Huashi Baidu decoction (Q-14) is a clinically approved formula for COVID-19 therapy with antiviral and anti-inflammatory effects. Here, an integrative pharmacological strategy was applied to identify the antiviral and anti-inflammatory bioactive compounds from Q-14. Overall, a total of 343 chemical compounds were initially characterized, and 60 prototype compounds in Q-14 were subsequently traced in plasma using ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Among the 60 compounds, six compounds (magnolol, glycyrrhisoflavone, licoisoflavone A, emodin, echinatin, and quercetin) were identified showing a dose-dependent inhibition effect on the SARS-CoV-2 infection, including two inhibitors (echinatin and quercetin) of the main protease (Mpro), as well as two inhibitors (glycyrrhisoflavone and licoisoflavone A) of the RNA-dependent RNA polymerase (RdRp). Meanwhile, three anti-inflammatory components, including licochalcone B, echinatin, and glycyrrhisoflavone, were identified in a SARS-CoV-2-infected inflammatory cell model. In addition, glycyrrhisoflavone and licoisoflavone A also displayed strong inhibitory activities against cAMP-specific 3',5'-cyclic phosphodiesterase 4 (PDE4). Crystal structures of PDE4 in complex with glycyrrhisoflavone or licoisoflavone A were determined at resolutions of 1.54 Å and 1.65 Å, respectively, and both compounds bind in the active site of PDE4 with similar interactions. These findings will greatly stimulate the study of TCMT-NDRD against COVID-19.

Strategy To Assess Zoonotic Potential Reveals Low Risk Posed by SARS-Related Coronaviruses from Bat and Pangolin.

In the last 2 decades, pathogens originating in animals may have triggered three coronavirus pandemics, including the coronavirus disease 2019 pandemic. Thus, evaluation of the spillover risk of animal severe acute respiratory syndrome (SARS)-related coronavirus (SARSr-CoV) is important in the context of future disease preparedness. However, there is no analytical framework to assess the spillover risk of SARSr-CoVs, which cannot be determined by sequence analysis alone. Here, we established an integrity framework to evaluate the spillover risk of an animal SARSr-CoV by testing how viruses break through key human immune barriers, including viral cell tropism, replication dynamics, interferon signaling, inflammation, and adaptive immune barriers, using human ex vivo lung tissues, human airway and nasal organoids, and human lung cells. Using this framework, we showed that the two pre-emergent animal SARSr-CoVs, bat BtCoV-WIV1 and pangolin PCoV-GX, shared similar cell tropism but exhibited less replicative fitness in the human nasal cavity or airway than did SARS-CoV-2. Furthermore, these viruses triggered fewer proinflammatory responses and less cell death, yet showed interferon antagonist activity and the ability to partially escape adaptive immune barriers to SARS-CoV-2. Collectively, these animal viruses did not fully adapt to spread or cause severe diseases, thus causing successful zoonoses in humans. We believe that this experimental framework provides a path to identifying animal coronaviruses with the potential to cause future zoonoses. IMPORTANCE Evaluation of the zoonotic risk of animal SARSr-CoVs is important for future disease preparedness. However, there are misconceptions regarding the risk of animal viruses. For example, an animal SARSr-CoV could readily infect humans. Alternately, human receptor usage may result in spillover risk. Here, we established an analytical framework to assess the zoonotic risk of SARSr-CoV by testing a series of virus-host interaction profiles. Our data showed that the pre-emergent bat BtCoV-WIV1 and pangolin PCoV-GX were less adapted to humans than SARS-CoV-2 was, suggesting that it may be extremely rare for animal SARSr-CoVs to break all bottlenecks and cause successful zoonoses.

SARS-CoV-2 Z-RNA activates the ZBP1-RIPK3 pathway to promote virus-induced inflammatory responses.

SARS-CoV-2 infection can trigger strong inflammatory responses and cause severe lung damage in COVID-19 patients with critical illness. However, the molecular mechanisms by which the infection induces excessive inflammatory responses are not fully understood. Here, we report that SARS-CoV-2 infection results in the formation of viral Z-RNA in the cytoplasm of infected cells and thereby activates the ZBP1-RIPK3 pathway. Pharmacological inhibition of RIPK3 by GSK872 or genetic deletion of MLKL reduced SARS-CoV-2-induced IL-1ß release. ZBP1 or RIPK3 deficiency leads to reduced production of both inflammatory cytokines and chemokines during SARS-CoV-2 infection both in vitro and in vivo. Furthermore, deletion of ZBP1 or RIPK3 alleviated SARS-CoV-2 infection-induced immune cell infiltration and lung damage in infected mouse models. These results suggest that the ZBP1-RIPK3 pathway plays a critical role in SARS-CoV-2-induced inflammatory responses and lung damage. Our study provides novel insights into how SARS-CoV-2 infection triggers inflammatory responses and lung pathology, and implicates the therapeutic potential of targeting ZBP1-RIPK3 axis in treating COVID-19.

ACE2-independent infection of T lymphocytes by SARS-CoV-2.

SARS-CoV-2 induced marked lymphopenia in severe patients with COVID-19. However, whether lymphocytes are targets of viral infection is yet to be determined, although SARS-CoV-2 RNA or antigen has been identified in T cells from patients. Here, we confirmed that SARS-CoV-2 viral antigen could be detected in patient peripheral blood cells (PBCs) or postmortem lung T cells, and the infectious virus could also be detected from viral antigen-positive PBCs. We next prove that SARS-CoV-2 infects T lymphocytes, preferably activated CD4 + T cells in vitro. Upon infection, viral RNA, subgenomic RNA, viral protein or viral particle can be detected in the T cells. Furthermore, we show that the infection is spike-ACE2/TMPRSS2-independent through using ACE2 knockdown or receptor blocking experiments. Next, we demonstrate that viral antigen-positive T cells from patient undergone pronounced apoptosis. In vitro infection of T cells induced cell death that is likely in mitochondria ROS-HIF-1a-dependent pathways. Finally, we demonstrated that LFA-1, the protein exclusively expresses in multiple leukocytes, is more likely the entry molecule that mediated SARS-CoV-2 infection in T cells, compared to a list of other known receptors. Collectively, this work confirmed a SARS-CoV-2 infection of T cells, in a spike-ACE2-independent manner, which shed novel insights into the underlying mechanisms of SARS-CoV-2-induced lymphopenia in COVID-19 patients.

Antibody-Dependent Enhancement of SARS-CoV-2 Infection of Human Immune Cells: In Vitro Assessment Provides Insight in COVID-19 Pathogenesis.

Patients with COVID-19 generally raise antibodies against SARS-CoV-2 following infection, and the antibody level is positively correlated to the severity of disease. Whether the viral antibodies exacerbate COVID-19 through antibody-dependent enhancement (ADE) is still not fully understood. Here, we conducted in vitro assessment of whether convalescent serum enhanced SARS-CoV-2 infection or induced excessive immune responses in immune cells. Our data revealed that SARS-CoV-2 infection of primary B cells, macrophages and monocytes, which express variable levels of FcγR, could be enhanced by convalescent serum from COVID-19 patients. We also determined the factors associated with ADE, and found which showed a time-dependent but not viral-dose dependent manner. Furthermore, the ADE effect is not associated with the neutralizing titer or RBD antibody level when testing serum samples collected from different patients. However, it is higher in a medium level than low or high dilutions in a given sample that showed ADE effect, which is similar to dengue. Finally, we demonstrated more viral genes or dysregulated host immune gene expression under ADE conditions compared to the no-serum infection group. Collectively, our study provides insight into the understanding of an association of high viral antibody titer and severe lung pathology in severe patients with COVID-19.

Screening of a Small Molecule Compound Library Identifies Toosendanin as an Inhibitor Against Bunyavirus and SARS-CoV-2.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus causing serious infectious disease with a high case-fatality of up to 50% in severe cases. Currently, no effective drug has been approved for the treatment of SFTSV infection. Here, we performed a high-throughput screening of a natural extracts library for compounds with activities against SFTSV infection. Three hit compounds, notoginsenoside Ft1, punicalin, and toosendanin were identified for displaying high anti-SFTSV efficacy, in which, toosendanin showed the highest inhibition potency. Mechanistic investigation indicated that toosendanin inhibited SFTSV infection at the step of virus internalization. The anti-viral effect of toosendanin against SFTSV was further verified in mouse infection models, and the treatment with toosendanin significantly reduced viral load and histopathological changes in vivo. The antiviral activity of toosendanin was further expanded to another bunyavirus and the emerging SARS-CoV-2. This study revealed a broad anti-viral effect of toosendanin and indicated its potential to be developed as an anti-viral drug for clinical use.

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010-2020: What Happened Before and After the COVID-19 Outbreak?

INTRODUCTION: Since the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19. METHODS: We conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1-3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children's Hospital of Fudan University during 2010-2020 were analyzed. RESULTS: Of the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September. CONCLUSIONS: These eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.

Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-19 patients.

Antibody-dependent cellular cytotoxicity (ADCC) responses to viral infection are a form of antibody regulated immune responses mediated through the Fc fragment. Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered ADCC responses contributes to COVID-19 disease development is currently not well understood. To understand the potential correlation between ADCC responses and COVID-19 disease development, we analyzed the ADCC activity and neutralizing antibody response in 255 individuals ranging from asymptomatic to fatal infections over 1 year post disease. ADCC was elicited by 10 days post-infection, peaked by 11-20 days, and remained detectable until 400 days post-infection. In general, patients with severe disease had higher ADCC activities. Notably, patients who had severe disease and recovered had higher ADCC activities than patients who had severe disease and deceased. Importantly, ADCC activities were mediated by a diversity of epitopes in SARS-COV-2-infected mice and induced to comparable levels against SARS-CoV-2 variants of concern (VOCs) (B.1.1.7, B.1.351, and P.1) as that against the D614G mutant in human patients and vaccinated mice. Our study indicates anti-SARS-CoV-2 ADCC as a major trait of COVID-19 patients with various conditions, which can be applied to estimate the extra-neutralization level against COVID-19, especially lethal COVID-19.

Viral dynamics and antibody responses in people with asymptomatic SARS-CoV-2 infection.

Over 40% of the coronavirus disease 2019 (COVID-19) COVID-19 patients were asymptomatically infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the immune responses of these asymptomatic individuals is a critical factor for developing the strategy to contain the COVID-19 pandemic. Here, we determined the viral dynamics and antibody responses among 143 asymptomatic individuals identified in a massive screening of more than 5 million people in eight districts of Wuhan in May 2020. Asymptomatic individuals were admitted to the government-designated centralized sites in accordance with policy. The incidence rate of asymptomatic infection is ~2.92/100,000. These individuals had low viral copy numbers (peaked at 315 copies/mL) and short-lived antibody responses with the estimated diminish time of 69 days. The antibody responses in individuals with persistent SARS-CoV-2 infection is much longer with the estimated diminish time of 257 days. These results imply that the immune responses in the asymptomatic individuals are not potent enough for preventing SARS-CoV-2 re-infection, which has recently been reported in recovered COVID-19 patients. This casts doubt on the efficacy of forming "herd-immunity" through natural SARS-CoV-2 infection and urges for the development of safe and effective vaccines.

Author Correction: Calcium channel blocker amlodipine besylate therapy is associated with reduced case fatality rate of COVID-19 patients with hypertension.

A Correction to this paper has been published: https://doi.org/10.1038/s41421-021-00267-0.

Calcium channel blocker amlodipine besylate therapy is associated with reduced case fatality rate of COVID-19 patients with hypertension.

The coronavirus disease (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread to >200 countries posing a global public health concern. Patients with comorbidity, such as hypertension suffer more severe infection with elevated mortality. The development of effective antiviral drugs is in urgent need to treat COVID-19 patients. Here, we report that calcium channel blockers (CCBs), a type of antihypertensive drug that is widely used in clinics, inhibited the post-entry replication events of SARS-CoV-2 in vitro, while no in vitro anti-SARS-CoV-2 effect was observed for the two other major types of antihypertensive drugs, namely, angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers. CCB combined with chloroquine showed a significantly enhanced anti-SARS-CoV-2 efficacy. A retrospective clinical investigation on hospitalized COVID-19 patients with hypertension as the only comorbidity revealed that the CCB amlodipine besylate therapy was associated with a decreased case fatality rate. The results from this study suggest that CCB administration to COVID-19 patients with hypertension as the comorbidity might improve the disease outcome.

SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to respiratory illness and multi-organ failure in critically ill patients. Although the virus-induced lung damage and inflammatory cytokine storm are believed to be directly associated with coronavirus disease 2019 (COVID-19) clinical manifestations, the underlying mechanisms of virus-triggered inflammatory responses are currently unknown. Here we report that SARS-CoV-2 infection activates caspase-8 to trigger cell apoptosis and inflammatory cytokine processing in the lung epithelial cells. The processed inflammatory cytokines are released through the virus-induced necroptosis pathway. Virus-induced apoptosis, necroptosis, and inflammation activation were also observed in the lung sections of SARS-CoV-2-infected HFH4-hACE2 transgenic mouse model, a valid model for studying SARS-CoV-2 pathogenesis. Furthermore, analysis of the postmortem lung sections of fatal COVID-19 patients revealed not only apoptosis and necroptosis but also massive inflammatory cell infiltration, necrotic cell debris, and pulmonary interstitial fibrosis, typical of immune pathogenesis in the lung. The SARS-CoV-2 infection triggered a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients. These discoveries might assist the development of therapeutic strategies to treat COVID-19.