Aglycone polyether ionophores as broad-spectrum agents inhibit multiple enveloped viruses including SARS-CoV-2 in vitro and successfully cure JEV infected mice (preprint)

Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo. Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.

Analysis of 8 274 cases of new coronavirus nucleic acid detection and co-infection in Wuhan / 中华检验医学杂志

Objective@#To investigate the positive rate for 2019-nCoV tests and co-infections in Wuhan district.@*Methods@#A total of 8 274 cases in Wuhan were enrolled in this cross-sectional study during January 20 to February 9, 2020, and were tested for 2019-nCoV using fluorescence quantitative PCR. Both respiratory tract samples (nasopharynx, oropharynx, sputum and alveolar lavage fluid) and non-respiratory tract samples (urine, feces, anal swabs, blood and conjunctival sac swabs) were collected. If both orf1ab and N genes are positive, they are classified as nucleic acid test positive group; if both orf1ab and N genes are negative, they are classified as negative group; if single gene target is positive, they are classified as suspicious group. Individuals were divided into male group and female group according to sex. At the same time, 316 patients were tested for 13 respiratory pathogens by multiplex PCR.@*Results@#Among the 8 274 subjects, 2 745 (33.2%) were 2019-nCoV infected; 5 277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test; and 252 cases (3.05%) was not definitive (inconclusive result). The age of cases with COVID-19 patients and inconclusive cases was significantly higher than that of cases without 2019-nCoV infection (40 vs 56, t=27.569, P<0.001; 52 vs 56, t=6.774, P<0.001). The positive rate of 13 respiratory pathogens multiple tests was significantly lower in 104 subjects who were positive for 2019-nCoV compared with those in subjects who were negative for 2019-nCoV test (5.77% vs 18.39%, χ2=24.105, P=0.003). Four types of respiratory tract samples and five types of non-respiratory tract samples were found to be positive for 2019-nCoV nucleic acid test.@*Conclusion@#The 2019-nCoV nucleic acid positive rate in male is higher than in female. Co-infections should be pay close attention in COVID-19 patients. 2019-nCoV nucleic acid can be detected in non-respiratory tract samples.

Immunodepletion with Hypoxemia: A Potential High Risk Subtype of Coronavirus Disease 2019 (preprint)

Background The outbreak of COVID-2019 is becoming a global public health emergency. Although its basic clinical features have been reported, the dynamic characteristics of immune system in COVID-2019 patients, especially those critical patients with refractory hypoxemia, are not yet well understood. We aim to describe the dynamic characteristics of immune system in 3 critical patients with refractory hypoxemia, and discuss the relationship between hypoxemia severity and immune cell levels, and the changes of gut microbes of COVID-2019 patient. Methods This is a retrospective study from 3 patients with 2019-nCoV infection admitted to Renmin Hospital of Wuhan University, a COVID-2019 designated hospital in Wuhan, from January 31 to February 6, 2020. All patients were diagnosed and classified based on the Diagnosis and Treatment of New Coronavirus Pneumonia (6th edition) published by the National Health Commission of China4. We recorded the epidemiological history, demographic features, clinical characteristics, symptoms and signs, treatment and clinical outcome in detail. Blood samples were collected and we determined the expression levels of immune cells (CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD16+56+ NK cells) in different time points. Nanopore Targeted Sequencing was used to determine the alterations of gut microbiota homeostasis. Results Apart from the clinical features described previously4, we found that four patients had decreased immune cells and refractory hypoxemia during the hospitalization, and the severity of hypoxemia was strongly correlated to the expression levels of immune cells. Additionally, we found that the proportion of probiotics was significantly reduced, such as Bifidobacterium, Lactobacillus, and Eubacterium, and the proportion of conditioned pathogenic bacteria was significantly increased, such as Corynebacterium of Actinobacteria and Ruthenibacterium of Firmicutes. Notably, all patients died. Conclusions We discussed the dynamic characteristics of host immune system and the imbalance of gut microbiota in 3 critical patients with COVID-2019. Hypoxemia severity was closely related with host immune cell levels, and the vicious circle between immune disorder and gut microbiota imbalance may be a high risk of fatal pneumonia. To the best of our knowledge, this is the first study which revealing that immunodepletion with refractory hypoxemia is a potential high risk subtype of COVID-2019 and the vicious circle between immune disorder and gut dysbiosis may be a high risk of fatal pneumonia.

Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses (preprint)

The ongoing novel coronavirus pneumonia COVID-19 outbreak in Wuhan, China, has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, current recommended methods exhibit high false-negative rates, low sensitivity, and cannot identify other respiratory virus infections, thereby resulting patient misdiagnosis and impeding epidemic containment. Combining the advantages of target amplification and long-read, real-time nanopore sequencing, we developed nanopore target sequencing (NTS) to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h. Parallel testing with approved qPCR kits of SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases confirmed that NTS identified more infected patients as positive, and could also monitor for mutated nucleic acid sequence or other respiratory virus infection in the test sample. NTS is thus suitable for contemporary COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses or pathogens.

Clinical diagnosis of 8274 samples with 2019-novel coronavirus in Wuhan (preprint)

Background: 2019-Novel coronavirus (2019-nCoV) outbreaks create challenges for hospital laboratories because thousands of samples must be evaluated each day. Sample types, interpretation methods, and corresponding laboratory standards must be established. The possibility of other infections should be assessed to provide a basis for clinical classification, isolation, and treatment. Accordingly, in the present study, we evaluated the testing methods for 2019-nCoV and co-infections. Methods: We used a fluorescence-based quantitative PCR kit urgently distributed by the Chinese CDC to detect 8274 close contacts in the Wuhan region against two loci on the 2019-nCoV genome. We also analyzed 613 patients with fever who underwent multiple tests for 13 respiratory pathogens; 316 subjects were also tested for 2019-nCoV. Findings: Among the 8274 subjects, 2745 (33.2%) had 2019-nCoV infection; 5277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test (non-019-nCoV); and 252 cases (3.0%) because only one target was positive, the diagnosis was not definitive. Sixteen patients who originally had only one positive target were re-examined a few days later; 14 patients (87.5%) were finally defined as 2019-nCoV-positive, and 2 (12.5%) were finally defined as negative. The positive rates of nCoV-NP and nCovORF1ab were 34.7% and 34.7%, respectively. nCoV-NP-positive only and nCovORF1ab-positive cases accounted for 1.5% and 1.5%, respectively. In the 316 patients with multiple respiratory pathogens, 104 were positive for 2019-nCov and 6/104 had co-infection with coronavirus (3/104), influenza A virus (2/104), rhinovirus (2/104), and influenza A H3N2 (1/104); the remaining 212 patients had influenza A virus (11/202), influenza A H3N2 (11/202), rhinovirus (10/202), respiratory syncytial virus (7/202), influenza B virus (6/202), metapneumovirus (4/202), and coronavirus (2/202). Interpretation: Clinical testing methods for 2019-nCoV require improvement. Importantly, 5.8% of 2019-nCoV infected and 18.4% of non-2019-nCoV-infected patients had other pathogen infections. It is important to treat combined infections and perform rapid screening to avoid cross-contamination of patients. A test that quickly and simultaneously screens as many pathogens as possible is needed.