Role of LL-37 in thrombotic complications in patients with COVID-19.

Blood clot formation induced by dysfunctional coagulation is a frequent complication of coronavirus disease 2019 (COVID-19) and a high-risk factor for severe illness and death. Neutrophil extracellular traps (NETs) are implicated in COVID-19-induced immunothrombosis. Furthermore, human cathelicidin, a NET component, can perturb the interaction between the SARS-CoV-2 spike protein and its ACE2 receptor, which mediates viral entry into cells. At present, however, the levels of cathelicidin antimicrobial peptides after SARS-CoV-2 infection and their role in COVID-19 thrombosis formation remain unclear. In the current study, we analyzed coagulation function and found a decrease in thrombin time but an increase in fibrinogen level, prothrombin time, and activated partial thromboplastin time in COVID-19 patients. In addition, the cathelicidin antimicrobial peptide LL-37 was upregulated by the spike protein and significantly elevated in the plasma of patients. Furthermore, LL-37 levels were negatively correlated with thrombin time but positively correlated with fibrinogen level. In addition to platelet activation, cathelicidin peptides enhanced the activity of coagulation factors, such as factor Xa (FXa) and thrombin, which may induce hypercoagulation in diseases with high cathelicidin peptide levels. Injection of cathelicidin peptides promoted the formation of thrombosis, whereas deletion of cathelicidin inhibited thrombosis in vivo. These results suggest that cathelicidin antimicrobial peptide LL-37 is elevated during SARS-CoV-2 infection, which may induce hypercoagulation in COVID-19 patients by activating coagulation factors.

SARS-CoV-2 Quasispecies Provides an Advantage Mutation Pool for the Epidemic Variants.

The dynamics of quasispecies afford RNA viruses a great fitness on cell tropism and host range. To study the quasispecies features and the intra-host evolution of SARS-CoV-2, we collected nine confirmed patients and sequenced the haplotypes of spike gene using a single-molecule real-time platform. Fourteen samples were extracted from sputum, nasopharyngeal swabs, or stool, which in total produced 283,655 high-quality circular consensus sequences. We observed a stable quasispecies structure that one master mutant (mean abundance ∼0.70), followed by numerous minor mutants (mean abundance ∼1.21 × 10-3). Under high selective pressure, minor mutants may obtain a fitness advantage and become the master ones. The later predominant substitution D614G existed in the minor mutants of more than one early patient. An epidemic variant had a possibility to be independently originated from multiple hosts. The mutant spectrums covered ∼85% amino acid variations of public genomes (GISAID; frequency ≥ 0.1) and likely provided an advantage mutation pool for the current/future epidemic variants. Notably, 32 of 35 collected antibody escape substitutions were preexistent in the early quasispecies. Virus populations in different tissues/organs revealed potentially independent replications. The quasispecies complexity of sputum samples was significantly lower than that of nasopharyngeal swabs (P = 0.02). Evolution analysis revealed that three continuous S2 domains (HR1, CH, and CD) had undergone a positive selection. Cell fusion-related domains may play a crucial role in adapting to the intrahost immune system. Our findings suggested that future epidemiologic investigations and clinical interventions should consider the quasispecies information that has missed by routine single consensus genome. IMPORTANCE RNA virus population in a host does not consist of a consensus single haplotype but rather an ensemble of related sequences termed quasispecies. The dynamics of quasispecies afford SARS-CoV-2 a great ability on genetic fitness during intrahost evolution. The process is likely achieved by changing the genetic characteristics of key functional genes, such as the spike glycoprotein. Previous studies have applied the next-generation sequencing (NGS) technology to evaluate the quasispecies of SARS-CoV-2, and results indicated a low genetic diversity of the spike gene. However, the NGS platform cannot directly obtain the full haplotypes without assembling, and it is also difficult to predict the extremely low-frequency variations. Therefore, we introduced a single-molecule real-time technology to directly obtain the haplotypes of the RNA population and further study the quasispecies features and intrahost evolution of the spike gene.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

Effect of SARS-CoV-2 coinfection was not apparent on the dynamics of chronic hepatitis B infection.

In patients coinfected with SARS-CoV-2 and HBV, liver injury was common. However, the interactions between SARS-CoV-2 and HBV coinfection remained unknown. Sixty-seven COVID-19 patients from the previous cohort were enrolled and classified into 2 groups (7 with HBsAg+ and 60 with HBsAg-). The association of HBV- and SARS-CoV-2-related markers were analyzed. During the acute course of SARS-CoV-2 infection, markers of HBV replication did not extensively fluctuate during SARS-CoV-2 infection. Coinfection with HBV did not extend the viral shedding cycle or incubation periods of SARS-CoV-2. Effects of SARS-CoV-2 on the dynamics of chronic HBV infection seemed not apparent. SARS-CoV-2 infection would not be the source of HBV reactivation in these individuals.

Shorter incubation period is associated with severe disease progression in patients with COVID-19.

The diagnosed COVID-19 cases revealed that the incubation periods (IP) varied a lot among patients. However, few studies had emphasized on the different clinical features and prognosis of patients with different IP. A total of 330 patients with laboratory-confirmed COVID-19 were enrolled and classified into immediate onset group(IP<3 days, I group, 57 cases) and late onset group(IP>10 days, L group, 75 cases) based on IP. The difference of clinical characteristics and prognosis of the two groups were compared. There were more patients with fever in I group than in L group(P = 0.003), and counts of all the total lymphocytes, total T lymphocytes, CD4 + and CD8 + T lymphocytes were significantly different between the two groups(all P < 0.01). Besides, patients in L group had more GGOs in CT scan than I group and there were more patients in I group receiving antibiotic treatment than in L group(P < 0.001). For disease aggravation, the median CT scores were comparable between the two groups, but individually, there were more patients with increased CT score during hospitalization in I group than in L group. The aggravation incidence of CT presentation was 21.1% in I group, significantly higher than L group(8.0%, P = 0.042). Multivariable COX models suggested that IP was the only independent factors for CT aggravation. Conclusively, patients with different IP were different in clinical symptoms, laboratory tests, and CT presentations. Shorter IP was associated with the aggravation of lung involvement in CT scan.

Accuracy of a nucleocapsid protein antigen rapid test in the diagnosis of SARS-CoV-2 infection.

OBJECTIVES: Rapid, reliable and easy-to-implement diagnostics that can be adapted in early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis are critical to combat the epidemic. SARS-CoV-2 nucleocapsid protein (NP) is an ideal target for viral antigen-based detection. A rapid and convenient method was developed based on fluorescence immunochromatographic (FIC) assay to detect the SARS-CoV-2 NP antigen. However, the accuracy of this diagnostic method needs to be examined. METHODS: This prospective study was carried out between 10 and 15 February 2020 in seven hospitals in Wuhan and one hospital in Chongqing, China. Participants with clinically suspected SARS-CoV-2 infection were enrolled. NP antigen testing by FIC assay and nucleic acid (NA) testing by real-time reverse transcriptase PCR (RT-PCR) were performed simultaneously in a blinded manner with the same nasopharyngeal swab sample. The diagnostic accuracy of NP antigen testing was calculated by taking NA testing of RT-PCR as the reference standard, in which samples with a cycle threshold (Ct) value of ≤40 were interpreted as positive for SARS-CoV-2. RESULTS: A total of 253 participants were enrolled; two participants were excluded from the analyses because of invalid NP testing results. Of 251 participants (99.2%) included in the diagnostic accuracy analysis, 201 (80.1%) had a Ct value of ≤40. With Ct value 40 as the cutoff of NA testing, the sensitivity, specificity and percentage agreement of the FIC assay was 75.6% (95% confidence interval, 69.0-81.3), 100% (95% confidence interval, 91.1-100) and 80.5% (95% confidence interval, 75.1-84.9) respectively. CONCLUSIONS: With RT-PCR assay as the reference standard, NP antigen testing by FIC assay shows high specificity and relatively high sensitivity in SARS-CoV-2 diagnosis in the early phase of infection.

TWIRLS, a knowledge-mining technology, suggests a possible mechanism for the pathological changes in the human host after coronavirus infection via ACE2.

Faced with the current large-scale public health emergency, collecting, sorting, and analyzing biomedical information related to the "SARS-CoV-2" should be done as quickly as possible to gain a global perspective, which is a basic requirement for strengthening epidemic control capacity. However, for human researchers studying viruses and hosts, the vast amount of information available cannot be processed effectively and in a timely manner, particularly if our scientific understanding is also limited, which further lowers the information processing efficiency. We present TWIRLS (Topic-wise inference engine of massive biomedical literatures), a method that can deal with various scientific problems, such as liver cancer, acute myeloid leukemia, and so forth, which can automatically acquire, organize, and classify information. Additionally, this information can be combined with independent functional data sources to build an inference system via a machine-based approach, which can provide relevant knowledge to help human researchers quickly establish subject cognition and to make more effective decisions. Using TWIRLS, we automatically analyzed more than three million words in more than 14,000 literature articles in only 4 hr. We found that an important regulatory factor angiotensin-converting enzyme 2 (ACE2) may be involved in host pathological changes on binding to the coronavirus after infection. On triggering functional changes in ACE2/AT2R, the cytokine homeostasis regulation axis becomes imbalanced via the Renin-Angiotensin System and IP-10, leading to a cytokine storm. Through a preliminary analysis of blood indices of COVID-19 patients with a history of hypertension, we found that non-ARB (Angiotensin II receptor blockers) users had more symptoms of severe illness than ARB users. This suggests ARBs could potentially be used to treat acute lung injury caused by coronavirus infection.