One viral sequence for each host? - The neglected within-host diversity as the main stage of SARS-CoV-2 evolution (preprint)

The standard practice of presenting one viral sequence for each infected individual implicitly assumes low within-host genetic diversity. It places the emphasis on the viral evolution between, rather than within, hosts. To determine this diversity, we collect SARS-CoV-2 samples from the same patient multiple times. Our own data in conjunction with previous reports show that two viral samples collected from the same individual are often very different due to the substantial within-host diversity. Each sample captures only a small part of the total diversity that is transiently and locally released from infected cells. Hence, the global SARS-CoV-2 population is a meta-population consisting of the viruses in all the infected hosts, each of which harboring a genetically diverse sub-population. Advantageous mutations must be present first as the within-host diversity before they are revealed as between-host polymorphism. The early detection of such diversity in multiple hosts could be an alarm for potentially dangerous mutations. In conclusion, the main forces of viral evolution, i.e., mutation, drift, recombination and selection, all operate within hosts and should be studied accordingly. Several significant implications are discussed.

Homo-harringtonine (HHT) - A highly effective drug against coronaviruses and the potential for large-scale clinical applications (preprint)

In the search for treatment schemes of COVID-19, we start by examining the general weakness of coronaviruses and then identify approved drugs attacking that weakness. The approach, if successful, should identify drugs with a specific mechanism that is at least as effective as the best drugs proposed and are ready for clinical trials. All coronaviruses translate their non-structural proteins (~16) in concatenation, resulting in a very large super-protein. Homo-harringtonine (HHT), which has been approved for the treatment of leukemia, blocks protein elongation very effectively. Hence, HHT can repress the replication of many coronaviruses at the nano-molar concentration. In two mouse models, HHT clears SARS-CoV-2 in 3 days, especially by nasal dripping of 40 ug per day. We also use dogs to confirm the safety of HHT delivered by nebulization. The nebulization scheme could be ready for large-scale applications at the onset of the next epidemics. For the current COVID-19, a clinical trial has been approved by the Ditan hospital of Beijing but could not be implemented for want of patients. The protocol is available to qualified medical facilities.

Intra-host dynamic variations in SARS-CoV-2 (preprint)

The mutations make uncertain to SARS-CoV-2 disease control and vaccine development. At population-level, single nucleotide polymorphism (SNPs) have displayed mutations for illustrating epidemiology, transmission, and pathogenesis of COVID-19. These mutations are to be expected by the analysis of intra-host level, which presented as intra-host variations (iSNVs). Here, we performed spatio-temporal analysis on iSNVs in 402 clinical samples from 170 patients, and observed an increase of genetic diversity along the day post symptom onset within individual patient and among subpopulations divided by gender, age, illness severity and viral shedding time, suggested a positive selection at intra-host level. The comparison of iSNVs and SNPs displayed that most of nonsynonymous mutations were not fixed suggested a purifying selection. This two-step fitness selection enforced iSNVs containing more nonsynonymous mutations, that highlight the potential characters of SARS-CoV-2 for viral infections and global transmissions.

Carboxypeptidase B Inhibits Over-activation of Anaphylatoxin-neutrophil Extracellular Trap Axis in COVID-19 Patients (preprint)

Background: Thrombosis and coagulopathy are highly prevalent in severe patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. Neutrophil extracellular traps (NETs) are primary components of immunothrombosis, whereas the mechanism of NET formation remains unclear. We aim to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19. Methods: : We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase-DNA. We detected complement-induced NET formation by immunofluorescent staining and evaluated the cytotoxicity to vascular endothelial HUVEC cells by CCK-8 assay. Results: : We found that the plasma levels of complements (C3 and C5) and NETs were closely related to coagulopathy and multiple organ dysfunction in patients with COVID-19. By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products. Conclusions: : Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

The strategies of perioperative management in orthopedic department during the pandemic of COVID-19 (preprint)

Background: Coronavirus disease 2019 (COVID-19) broken out and spread rapidly nationwide at the beginning of 2020, which has brought huge impacts to people and work. The current situation of prevention and control is severe and urge guidance for clinicians, especially for medical systems. In the hope of providing a reference and recommendation for the prevention and control of the COVID-19, we carried out research to improve the quality of patients care and prevention during this epidemic. Methods: : All of the staff were trained rapidly to master personal protection in our department. We reviewed the patients’ discharged records who underwent surgery in our department during January 1st to March 1st in 2019 and January 1st to March 1st in 2020. The managements of the surgery patients and flow charts were described and analyzed. Post-operation outcomes of the patients including duration, complications, surgical site infection (SSI), system infection, re-operation, and mortality. Both chi-squared test and Student’s t-test were performed to determine the relationship between the two periods in term of post-operation outcomes. Results: : Descriptive statistics analysis revealed that demographic of the patients between the two periods is similar. We had been benefited from the strict flow charts, smart robot and protection equipment in management of perioperative for orthopedic patients. With the help of the strict flow charts and smart equipment, post-operation outcomes of the patients revealed that the rates of the complications and re-operation had been reduced significantly ( p ＜0.05), while duration of operation, SSI and system infection had no significantly difference between two periods ( p ＞0.05). No patient and staff caught COVID-19 infection or mortality during the epidemic. Conclusions: : Our study indicated that medical quality and efficiency were affected little with the help of strategies described above during the epidemic, which could be a reference tool for medical staff in routine clinical practice for admission of patients around the world. What’s more, the provided strategies, which may evolve over time, could be used as empirical guidance and reference for orthopedic peers to get through the pandemic and ensure the normal operation of the hospital.

Impaired cellular immunity to SARS-CoV-2 in severe COVID-19 patients (preprint)

The World Health Organization has declared SARS-CoV-2 virus outbreak a world-wide pandemic. Individuals infected by the virus exhibited different degrees of symptoms, the basis of which remains largely unclear. Currently, though convalescent individuals have been shown with both cellular and humoral immune responses, there is very limited understanding on the immune responses, especially adaptive immune responses, in patients with severe COVID-19. Here, we examined 10 blood samples from COVID-19 patients with acute respiratory distress syndrome (ARDS). The majority of them (70%) mounted SARS-CoV-2-specific humoral immunity with production of neutralizing antibodies. However, compared to healthy controls, the percentages and absolute numbers of both NK cells and CD8+ T cells were significantly reduced, accompanied with decreased IFN{gamma} expression in CD4+ T cells in peripheral blood from severe patients. Most notably, we failed in detecting SARS-CoV-2-specific IFN{gamma} production by peripheral blood lymphocytes from these patients. Our work thus indicates that COVID-19 patients with severe symptoms are associated with defective cellular immunity, which not only provides insights on understanding the pathogenesis of COVID-19, but also has implications in developing an effective vaccine to SARS-CoV-2.

Olfactory and Gustatory Dysfunction as An Early Identifier of COVID-19 in Adults and Children: An International Multicenter Study (preprint)

Abstract Objective: Evaluate the prevalence and characteristics of olfactory or gustatory dysfunction in COVID-19 patients Study Design: Multicenter Case Series Setting: 5 tertiary care hospitals (3 in China, 1 in France, 1 in Germany) Subjects and Methods: 394 PCR confirmed COVID-19 positive patients were screened, and those with olfactory or gustatory dysfunction were included. Data including demographics, COVID-19 severity, patient outcome, and the incidence and degree of olfactory and/or gustatory dysfunction were collected and analyzed. The Questionnaire of Olfactory Disorders (QOD) and Visual Analogue Scale (VAS) were used to quantify olfactory and gustatory dysfunction respectively. All subjects at one hospital (Shanghai) without subjective olfactory complaints underwent objective testing. Results: Of 394 screened subjects, 161 (41%) reported olfactory and/or gustatory dysfunction and were included. Incidence of olfactory and/or gustatory disorders in Chinese (n=239), German (n=39) and French (n=116) cohorts were 32%, 69%, and 49% 138 respectively. The median age of included subjects was 39 years old, 92/161 (57%) were male, and 10/161 (6%) were children. Of included subjects, 10% had only olfactory or gustatory symptoms, and 19% had olfactory and/or gustatory complaints prior to any other COVID-19 symptom. Of subjects with objective olfactory testing, 10/90 demonstrated abnormal chemosensory function despite reporting normal subjective olfaction. 43% (44/102) of subjects with follow-up showed symptomatic improvement in olfaction or gustation. Conclusions: Olfactory and/or gustatory disorders may represent early or isolated symptoms of SARS-CoV-2 infection. They may serve as a useful additional screening criterion, particularly for the identification of patients in the early stages of infection.

MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples (preprint)

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for the understanding of viral spread and viral evolution, as well as for vaccine development. Existing sample preparation methods for viral genome sequencing are demanding on user technique and time, and thus not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We have developed MetagenomIc RNA EnRichment VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. This approach uses direct tagmentation of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library construction process, while subsequent targeted enrichment can generate viral genomes with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA is compatible with clinical nucleic extracts containing carrier RNA. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations during outbreaks, both current and future.

Broad and differential animal ACE2 receptor usage by SARS-CoV-2 (preprint)

The COVID-19 pandemic has caused an unprecedented global public health and economy crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2 from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activity, respectively. Among the remaining species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models. ImportanceSARS-CoV-2 uses human ACE2 as primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologues and found that wild type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models and molecular basis of receptor binding for SARS-CoV-2.

First Case of COVID-19 Complicated with Fulminant Myocarditis: A case Report and Insights (preprint)

Background: Coronavirus Disease 2019 (COVID-19) has been demonstrated to be the cause of pneumonia. Nevertheless, it has not been reported as the cause of acute myocarditis or fulminant myocarditis. Case presentation: A 63-year-old male was admitted with pneumonia and cardiac symptoms. He was genetically confirmed as having COVID-19 according to sputum testing on the day of admission. He also had elevated troponin I (Trop I) level (up to 11.37 g/L) and diffuse myocardial dyskinesia along with a decreased left ventricular ejection fraction (LVEF) on echocardiography. The highest level of interleukin-6 was 272.40 pg/ml. Bedside chest radiographs showed typical ground-glass changes indicative of viral pneumonia. Laboratory test results for viruses that cause myocarditis were all negative. The patient conformed to the diagnostic criteria of the Chinese expert consensus statement for fulminant myocarditis. After receiving antiviral therapy and mechanical life support, Trop I was reduced to 0.10 g/L, and interleukin-6 was reduced to 7.63 pg/ml. Moreover, the LVEF of the patient gradually recovered to 68%. The patient died of aggravation of secondary infection on the 33rd day of hospitalization. Conclusion: COVID-19 patients may develop severe cardiac complications such as myocarditis and heart failure. This is the first report of COVID-19 complicated with fulminant myocarditis. The mechanism of cardiac pathology caused by COVID-19 needs further study.

LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2 (preprint)

C3A is a sub-clone of human hepatoblastoma HepG2 cell line with the strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ADAP2, GILT and LY6E, three cellular proteins with known activity of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a distinct mechanism. ImportanceVirus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

First Case of COVID-19 Infection with Fulminant Myocarditis Complication: Case Report and Insights (preprint)

Background: The Coronavirus Disease 2019 (COVID-19) has been demonstrated as the cause of pneumonia. Nevertheless, it has not been reported as the cause of acute myocarditis or fulminant myocarditis. Case Presentation: A 63-year-old male was admitted with pneumonia and cardiac symptoms. He was genetically confirmed as COVID-19 by testing sputum on the first day of admission. He also had an elevated troponin-I (Trop I) level and diffuse myocardial dyskinesia along with decreased left ventricular ejection fraction (LVEF) on echocardiography. The highest level of Interleukin 6 was 272.40pg/ml. Bedside chest radiograph had typical ground-glass changes of viral pneumonia. The laboratory test results of virus that can cause myocarditis are all negative. The patient conformed to the diagnostic criteria of Chinese expert consensus statement for fulminant myocarditis. After receiving antiviral therapy and mechanical life support, the Trop I reduced to 0.10 g/L, and Interleukin 6 was 7.63 pg/ml. Meanwhile the LVEF of the patient gradually recovered to 68%. Conclusion: COVID-19 patients may develop severe cardiac complications such as myocarditis and heart failure, and this is the first case of COVID-19 infection complicated with fulminant myocarditis. The mechanism of cardiac pathology caused by COVID-19 needs further study.