The clinical and immunological characteristics of COVID-19 patients with delayed SARS-CoV-2 virus clearance.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a great threat to human health. Some severe COVID-19 patients still carried detectable levels of SARS-CoV-2 even after prolonged intensive care unit treatment. However, the immunological features of these COVID-19 patients with delayed virus clearance (CDVC) are still unclear. METHODS: We retrospectively reviewed the clinical and immunological data of 13 CDVC cases, who were admitted into one hospital in Wuhan from February to April 2020. These data were also compared to those of perished (n = 9) and recovered (n = 52) cases. The expression of the exhaustion marker PD-1 on circulating T cells of these patients was measured by flow cytometry. RESULTS: High levels of serum interleukin-6 (IL-6), IL-1ß, IL-8, as well as other inflammatory mediators, were seen in CDVC cases. Severe lymphopenia was observed in CDVC patients with the counts of total lymphocytes (0.9 × 109 /L), CD4+ T cells (0.35 × 109 /L), and CD8+ T cells (0.28 × 109 /L) below their corresponding lower limits of normal range. Similar to the perished group, CDVC cases have higher percentages of CD25+ Foxp3+ regulatory T cells (Treg) in circulation. Moreover, enhanced expression of the exhaustion marker PD-1 on CCR7- CD45RA+ effector, CCR7+ CD45RA- central memory, and CCR7- CD45RA- effector memory CD4+ and CD8+ T cells were also observed in CDVC cases. CONCLUSION: CDVC patients still have SARS-CoV-2 and these cases manifest with severe clinical symptoms due to persistent inflammation. Augmentation of the frequency of circulating Treg, severe lymphopenia, and functional exhaustion of T cells might lead to inefficient clearance of SARS-CoV-2. Therefore, enhancing lymphocyte counts and reversing T-cell exhaustion might be key methods to boost immune responses and eliminate SARS-CoV-2 in CDVC patients.

Changes in the IL-18, IL-22, and T lymphocyte subset levels in patients with hepatitis B-related liver cirrhosis and their predictive values for hepatorenal syndrome.

OBJECTIVE: To investigate the changes in the interleukin (IL)-18, IL-22, and T lymphocyte subset levels in patients with hepatitis B-related liver cirrhosis and to determine their predictive values for hepatorenal syndrome (HRS). METHODS: Clinical data of 70 healthy individuals (group A) and 84 patients with hepatitis B-related liver cirrhosis (group B) admitted to Hospital 989 of the PLA Joint Logistics Support Force were retrospectively collected. The serum levels of IL-18 and IL-22, concentrations of cluster of differentiation (CD)3+, CD4+, and CD8+ cells, as well as the CD4+/CD8+ ratio in the peripheral blood T lymphocyte subsets were measured. Further, their predictive values for HRS were determined. Logistic regression analysis was employed to identify independent risk factors for HRS. RESULTS: In group B, the posttreatment IL-18 and IL-22 levels and CD8+ cell concentration significantly decreased after treatment, whereas the CD3+ and CD4+ cell concentrations and CD4+/CD8+ ratio increased. Notably, the serum IL-18 and IL-22 levels were higher in patients with HRS than in those without. Also, the CD3+ and CD4+ cell concentrations and CD4+/CD8+ ratio in the peripheral blood were lower in patients with HRS than in those without. The sensitivities of the serum IL-18 and IL-22 levels for predicting HRS were 90.32% and 80.65%, and the specificities were 71.70% and 77.36%, respectively. The sensitivities of CD3+, CD4+, and CD8+ cell concentrations for predicting HRS were 77.42%, 90.32%, and 83.87%, and the specificity was 67.92%, 64.15%, and 52.83%, respectively. Moreover, the sensitivity and specificity of CD4+/CD8+ ratio for predicting HRS were 80.65% and 86.79%, respectively. CONCLUSIONS: IL-18, IL-22, and T lymphocyte subset levels may have significant implications in the progression of hepatitis B-related liver cirrhosis, and detecting these markers could aid in treatment, evaluation, and prediction of HRS in patients. Furthermore, IL-18 and IL-22 levels and the CD4+/CD8+ ratio were identified as independent risk factors for HRS.

Characteristics of lymphocyte subset alterations in COVID-19 patients with different levels of disease severity.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a respiratory disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which had rapidly spread all over the world and caused public health emergencies in the past two years. Although the diagnosis and treatment for COVID-19 have been well defined, the immune cell characteristics and the key lymphocytes subset alterations in COVID-19 patients have not been thoroughly investigated. METHODS: The levels of immune cells including T cells, B cells, and natural killer (NK) cells in 548 hospitalized COVID-19 patients, and 30 types of lymphocyte subsets in 125 hospitalized COVID-19 patients admitted to Wuhan Huoshenshan Hospital of China were measured using flow cytometry. The relationship between lymphocytes subsets with the cytokine interleukin-6 (IL-6) and the characteristics of lymphocyte subsets in single-cell RNA sequencing (scRNA-seq) data obtained from peripheral blood mononuclear cells (PBMCs) were also analysed in COVID-19 patients. RESULTS: In this study, we found that patients with critical COVID-19 infection exhibited an overall decline in lymphocytes including CD4+ T cells, CD8+ T cells, total T cells, B cells, and NK cells compared to mild and severe patients. However, the number of lymphocyte subsets, such as CD21low CD38low B cells, effector T4 cells, and PD1+ depleted T8 cells, was moderately increased in critical COVID-19 patients compared to mild cases. Notably, except for effector memory T4 cells, plasma blasts and Tregs, the number of all lymphocyte subsets was markedly decreased in COVID-19 patients with IL-6 levels over 30-fold higher than those in healthy cases. Moreover, scRNA-seq data showed obvious differences in the distribution and numbers of lymphocyte subsets between COVID-19 patients and healthy persons, and subsets-specific marker genes of lymphocyte subsets including CD4, CD19, CCR7, and IL7R, were markedly decreased in COVID-19 patients compared with those in healthy cases. CONCLUSION: A comprehensive decrease in immune cell and lymphocyte subsets in critical COVID-19 patients, and peripheral lymphocyte subset alterations showed a clear association with clinical characteristics.

The reference genome and organelle genomes of wasabi (Eutrema japoniacum).

Wasabi (Eutrema japonicum) is one of the most famous vegetable crops in the family Brassicaceae. However, a limited genomic resource is available, which hinders genomic breeding and understanding of the genetic basis of vital traits. Here, we generated the genome assembly of wasabi using the hybrid genome assembly strategy, which combined the Nanopore long reads and Illumina reads. The genome assembly contains 687M bp and 39,534 high-quality annotated gene models. Besides, we annotated 68.85% of the genomic sequences as repetitive elements, including 43.72% of retrotransposons and 18.99% of DNA transposons. Using the customized pipeline, we also generated the complete organelle genomes of wasabi. This reference genome could provide essential genomic resources for evolution, breeding, and exploring the unique biological traits of wasabi.

SARS-CoV-2 Induces Lymphocytopenia by Promoting Inflammation and Decimates Secondary Lymphoid Organs.

While lymphocytopenia is a common characteristic of coronavirus disease 2019 (COVID-19), the mechanisms responsible for this lymphocyte depletion are unclear. Here, we retrospectively reviewed the clinical and immunological data from 18 fatal COVID-19 cases, results showed that these patients had severe lymphocytopenia, together with high serum levels of inflammatory cytokines (IL-6, IL-8 and IL-10), and elevation of many other mediators in routine laboratory tests, including C-reactive protein, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase and natriuretic peptide type B. The spleens and hilar lymph nodes (LNs) from six additional COVID-19 patients with post-mortem examinations were also collected, histopathologic detection showed that both organs manifested severe tissue damage and lymphocyte apoptosis in these six cases. In situ hybridization assays illustrated that SARS-CoV-2 viral RNA accumulates in these tissues, and transmission electronic microscopy confirmed that coronavirus-like particles were visible in the LNs. SARS-CoV-2 Spike and Nucleocapsid protein (NP) accumulated in the spleens and LNs, and the NP antigen restricted in angiotensin-converting enzyme 2 (ACE2) positive macrophages and dendritic cells (DCs). Furthermore, SARS-CoV-2 triggered the transcription of Il6, Il8 and Il1b genes in infected primary macrophages and DCs in vitro, and SARS-CoV-2-NP+ macrophages and DCs also manifested high levels of IL-6 and IL-1ß, which might directly decimate human spleens and LNs and subsequently lead to lymphocytopenia in vivo. Collectively, these results demonstrated that SARS-CoV-2 induced lymphocytopenia by promoting systemic inflammation and direct neutralization in human spleen and LNs.

Upregulated IL-6 Indicates a Poor COVID-19 Prognosis: A Call for Tocilizumab and Convalescent Plasma Treatment.

A comprehensive understanding of the dynamic changes in interleukin-6 (IL-6) levels is essential for monitoring and treating patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). By analyzing the correlations between IL-6 levels and health conditions, underlying diseases, several key laboratory detection indices, and the prognosis of 1,473 patients with the coronavirus disease 2019 (COVID-19), the role of IL-6 during SARS-CoV-2 infection was demonstrated. Our results indicated that IL-6 levels were closely related to age, sex, body temperature, oxygen saturation (SpO2) of blood, and underlying diseases. As a stable indicator, the changes in IL-6 levels could indicate the inflammatory conditions during a viral infection. Two specific treatments, namely, tocilizumab and convalescent plasma therapy (CPT), decreased the level of IL-6 and relieved inflammation. CPT has an important role in the therapy for patients with critical COVID-19. We also found that patients with IL-6 levels, which were 30-fold higher than the normal level, had a poor prognosis compared to patients with lower levels of IL-6.

3044 Cases reveal important prognosis signatures of COVID-19 patients.

Critical patients and intensive care unit (ICU) patients are the main population of COVID-19 deaths. Therefore, establishing a reliable method is necessary for COVID-19 patients to distinguish patients who may have critical symptoms from other patients. In this retrospective study, we firstly evaluated the effects of 54 laboratory indicators on critical illness and death in 3044 COVID-19 patients from the Huoshenshan hospital in Wuhan, China. Secondly, we identify the eight most important prognostic indicators (neutrophil percentage, procalcitonin, neutrophil absolute value, C-reactive protein, albumin, interleukin-6, lymphocyte absolute value and myoglobin) by using the random forest algorithm, and find that dynamic changes of the eight prognostic indicators present significantly distinct within differently clinical severities. Thirdly, our study reveals that a model containing age and these eight prognostic indicators can accurately predict which patients may develop serious illness or death. Fourthly, our results demonstrate that different genders have different critical illness rates compared with different ages, in particular the mortality is more likely to be attributed to some key genes (e.g. ACE2, TMPRSS2 and FURIN) by combining the analysis of public lung single cells and bulk transcriptome data. Taken together, we urge that the prognostic model and first-hand clinical trial data generated in this study have important clinical practical significance for predicting and exploring the disease progression of COVID-19 patients.

Evolution of COVID-19 in patients with autoimmune rheumatic diseases.

The characteristics of COVID-19 patients with autoimmune rheumatic diseases (AIRD) have rarely been reported. Patients with AIRD have suppressed immune defense function, which may increase their susceptibility to COVID-19. However, the immunosuppressive agents AIRD patients routinely used may be beneficial for protecting the cytokine storm caused by SARS-CoV-2. In this retrospective study, we included all confirmed cases in Huoshenshan Hospital from February 4 to April 9. Data were extracted from electronic medical records and were analyzed for clinical and laboratory features using SPSS (version 25.0). Of 3059 patients, 21 had the comorbidities with systematic lupus erythematosus (SLE) and/or rheumatoid arthritis (RA), including 5 with SLE, 15 with RA, and 1 with Rhupus. The proportion was 57.1% for severe cases, 61.9% for either severe or critical cases, and 4.8% for critical cases. The main manifestations, ARDS and ICU admission rate, as well as the mortality and length of hospital stay of COVID-19 in AIRD patients were similar to COVID-19 patients in the general population. Our preliminary experience shows that patients with AIRD tend to have a higher risk of SARS-CoV-2 infection, and may be at risk for a severe but less likely critical disease course. Further investigation is needed to understand the immunological features of these diseases.

Dynamic changes in anti-SARS-CoV-2 antibodies during SARS-CoV-2 infection and recovery from COVID-19.

Deciphering the dynamic changes in antibodies against SARS-CoV-2 is essential for understanding the immune response in COVID-19 patients. Here we analyze the laboratory findings of 1,850 patients to describe the dynamic changes of the total antibody, spike protein (S)-, receptor-binding domain (RBD)-, and nucleoprotein (N)-specific immunoglobulin M (IgM) and G (IgG) levels during SARS-CoV-2 infection and recovery. The generation of S-, RBD-, and N-specific IgG occurs one week later in patients with severe/critical COVID-19 compared to patients with mild/moderate disease, while S- and RBD-specific IgG levels are 1.5-fold higher in severe/critical patients during hospitalization. The RBD-specific IgG levels are 4-fold higher in older patients than in younger patients during hospitalization. In addition, the S- and RBD-specific IgG levels are 2-fold higher in the recovered patients who are SARS-CoV-2 RNA negative than those who are RNA positive. Lower S-, RBD-, and N-specific IgG levels are associated with a lower lymphocyte percentage, higher neutrophil percentage, and a longer duration of viral shedding. Patients with low antibody levels on discharge might thereby have a high chance of being tested positive for SARS-CoV-2 RNA after recovery. Our study provides important information for COVID-19 diagnosis, treatment, and vaccine development.

Successful recovery of COVID-19-associated recurrent diarrhea and gastrointestinal hemorrhage using convalescent plasma.

BACKGROUND: Gastrointestinal symptoms are not rare among coronavirus disease 2019 (COVID-19) patients, but there have been no reports regarding convalescent plasma therapy for the recovery of gastrointestinal problems in COVID-19 patients. CASE PRESENTATION: We present two cases of patients with COVID-19-associated recurrent diarrhea and positive fecal occult blood who successfully recovered after a one-time convalescent plasma administration. CONCLUSION: When COVID-19 patients develop recurrent or refractory gastrointestinal symptoms and fail to respond to the available treatment, alternative therapy with convalescent plasma administration may be considered.

Characterization and variation of the rhizosphere fungal community structure of cultivated tetraploid cotton.

Rhizosphere fungal communities exert important influencing forces on plant growth and health. However, information on the dynamics of the rhizosphere fungal community structure of the worldwide economic crop cotton (Gossypium spp.) is limited. In the present study, next-generation sequencing of nuclear ribosomal internal transcribed spacer-1 (ITS1) was performed to characterize the rhizosphere fungal communities of G. hirsutum cv. TM-1 (upland cotton) and G. barbadense cv. Hai 7124 (island cotton). The plants were grown in field soil (FS) that had been continuously cropped with cotton and nutrient-rich soil (NS) that had not been cropped. The fungal species richness, diversity, and community composition were analyzed and compared among the soil resources, cotton genotypes, and developmental stages. We found that the fungal community structures were different between the rhizosphere and bulk soil and the difference were significantly varied between FS and NS. Our results suggested that cotton rhizosphere fungal community structure variation may have been primarily influenced by the interaction of cotton roots with different soil resources. We also found that the community composition of the cotton rhizosphere fungi varied significantly during different developmental stages. In addition, we observed fungi that was enriched or depleted at certain developmental stages and genotypes in FS and NS, and these insights can lay a foundation for deep research into the dynamics of pathogenic fungi and nutrient absorption of cotton roots. This research illustrates the characteristics of the cotton rhizosphere fungal communities and provides important information for understanding the potential influences of rhizosphere fungal communities on cotton growth and health.

The Variation in the Rhizosphere Microbiome of Cotton with Soil Type, Genotype and Developmental Stage.

Plant roots and soil microorganisms interact with each other mainly in the rhizosphere. Changes in the community structure of the rhizosphere microbiome are influenced by many factors. In this study, we determined the community structure of rhizosphere bacteria in cotton, and studied the variation of rhizosphere bacterial community structure in different soil types and developmental stages using TM-1, an upland cotton cultivar (Gossypium hirsutum L.) and Hai 7124, a sea island cotton cultivar (G. barbadense L.) by high-throughput sequencing technology. Six bacterial phyla were found dominantly in cotton rhizosphere bacterial community including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria, and Verrucomicrobia. The abundance of Acidobacteria, Cyanobacteria, Firmicutes, Planctomycetes and Proteobacteria were largely influenced by cotton root. Bacterial α-diversity in rhizosphere was lower than that of bulk soil in nutrient-rich soil, but higher in cotton continuous cropping field soil. The ß-diversity in nutrient-rich soil was greater than that in continuous cropping field soil. The community structure of the rhizosphere bacteria varied significantly during different developmental stages. Our results provided insights into the dynamics of cotton rhizosphere bacterial community and would facilitate to improve cotton growth and development through adjusting soil bacterial community structure artificially.

Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity.

Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors.

[Establishment and identification of human hepatocellular carcinoma line stably expressing hepatitis C virus core protein].

OBJECTIVE: To establish SMMC-7721 human hepatocellular carcinoma cell line stably expressing hepatitis C virus (HCV) core protein. METHODS: A lentiviral vector containing HCV core gene was constructed and transfected into HEK293T cells to package recombinant lentivirus (rLV-core) containing ZsGreen and HCV core genes. The SMMC-7721 cells were infected with the rLV-core. The expression of HCV core mRNA was examined by real-time fluorescent quantitative PCR and the HCV core protein was detected by immunofluorescence cytochemistry and Western blotting. The stably transfected cell line was screened. RESULTS: The lentiviral vector was confirmed by enzyme digestion and sequencing. The green fluorescence was seen under fluorescence microscope 48 hours after virus packaging. The SMMC-7721 cell line stably expressing HCV core protein was obtained after infected with the rLV-core. Real-time PCR showed the expression of HCV core mRNA, and both immunofluorescence cytochemistry and Western blotting verified the expression of HCV core protein. CONCLUSION: The SMMC-7721 human hepatocellular carcinoma cell line stably expressing HCV core protein has been established successfully.