Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models.

The outbreak of coronavirus disease 2019 (COVID-19) has resulted in a global pandemic due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of this manuscript's publication, remdesivir is the only COVID-19 treatment approved by the United States Food and Drug Administration. However, its effectiveness is still under question due to the results of the large Solidarity Trial conducted by the World Health Organization. Herein, we report that the parent nucleoside of remdesivir, GS-441524, potently inhibits the replication of SARS-CoV-2 in Vero E6 and other cell lines. Challenge studies in both an AAV-hACE2 mouse model of SARS-CoV-2 and in mice infected with murine hepatitis virus, a closely related coronavirus, showed that GS-441524 was highly efficacious in reducing the viral titers in CoV-infected organs without notable toxicity. Our results support that GS-441524 is a promising and inexpensive drug candidate for treating of COVID-19 and other CoV diseases.

Prevalence and Risk Factors of Anxious and Depressive Symptoms in First-trimester Females and Their Partners: a study in the post-pandemic era of COVID-19 in China (preprint)

Background: The pandemic of coronavirus disease 2019 lastingly affects public mental health. Many studies have described symptoms of anxiety and depression in pregnant women during the pandemic. However, limited study focuses on the prevalence and risk factors of mood symptoms among females and their partners during early pregnancy in the post-pandemic era in China, which was the aim of the study and could promote clinical attention and suggest possible directions for intervention. Methods: One hundred and sixty-nine first-trimester couples were enrolled. The Edinburgh Postnatal Depression Scale, Patient Health Questionnaire-9, Generalized Anxiety Disorder 7-Item, Family Assessment Device-General Functioning (FAD-GF), and Quality of Life Enjoyment and Satisfaction Questionnaire, Short Form (Q-LES-Q-SF) were applied. Data were mainly analyzed through the binominal logistic regression analysis. Results: 17.8% and 5.9% of first-trimester females had depressive and anxious symptoms, respectively. Among partners, 12.4% and 9.5% had depressive and anxious symptoms, respectively. In females, higher scores of FAD-GF (OR= 5.461 and 14.759; P< 0.05) and lower scores of Q-LES-Q-SF (OR= 0.830 and 0.715; P< 0.01) were related to the risk of depressive and anxious symptoms. A history of smoking and higher scores of FAD-GF were associated with the risk of depressive and anxious symptoms in partners (OR = 4.906 and 6.885; P< 0.05). Conclusions: This study prompted still prominent mood symptoms in the post-pandemic era. Family functioning, quality of life, or a smoking history increased risks of mood symptoms among early pregnant families, which might facilitate the updating of medical intervention. However, the current study did not further explore interventions based on these findings.

The changes in psychological symptoms of COVID-19 patients after “re-positive”

Background Previous studies have showed that individuals infected with COVID-19 were more likely to report psychological symptoms. However, little is known about the changes from testing positive to negative to positive again. Methods This survey was conducted through the questionnaires including the 7-item Generalized Anxiety Disorder (GAD-7), the 9-item Patient Health Questionnaire (PHQ-9), as well as the Self-Rating Scale of Sleep (SRSS) to explore the psychological status of COVID-19 and re-positive cases. ″re-positive″ is defined as a positive RT-PCR test at any time during the recovery period after testing negative. Results A total of 94 COVID-19 patients presented the prevalence rates of anxiety, depression, insomnia, and any of the three psychological symptoms being 26.6, 8.6, 12.8, and 31.9%, respectively. Among these, 32 cases were re-tested positive during the recovery period, with the prevalence rates of anxiety, depression, insomnia, and any of the three psychological symptoms being 21.9, 18.7, 31.2, and 37.5%, respectively. The psychological status after re-positive showed a significant decrease in anxiety (P = 0.023), an increase in depression, and a significant rise in insomnia (P = 0.035). For those with no psychological symptoms during initial-positive, after re-positive, 5.88% reported anxiety, 5.88% reported depression, and 11.76% reported insomnia. For those who experienced only anxiety symptoms during initial-positive, after re-positive, 33.3% reported depression, and 33.3% reported insomnia. Conclusions Our findings encompassed the urgent concern for anxiety in initial-positive COVID-19 patients, depression in re-positive COVID-19 patients, and insomnia in both initial and re-positive patients, hence enabling targeted interventions for appeasing the psychological burden of COVID-19 patients.

Evidence of Infection of Human Embryonic Stem Cells by SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially described to target the respiratory system and now has been reported to infect a variety of cell types, including cardiomyocytes, neurons, hepatocytes, and gut enterocytes. However, it remains unclear whether the virus can directly infect human embryonic stem cells (hESCs) or early embryos. Herein, we sought to investigate this question in a cell-culture system of hESCs. Both the RNA and S protein of SARS-CoV-2 were detected in the infected hESCs and the formation of syncytium was observed. The increased level of subgenomic viral RNA and the presence of dsRNA indicate active replication of SARS-CoV-2 in hESCs. The increase of viral titers in the supernatants revealed virion release, further indicating the successful life cycle of SARS-CoV-2 in hESCs. Remarkably, immunofluorescence microscopy showed that only a small portion of hESCs were infected, which may reflect low expression of SARS-CoV-2 receptors. By setting |log2 (fold change)| > 0.5 as the threshold, a total of 1,566 genes were differentially expressed in SARS-CoV-2-infected hESCs, among which 17 interferon-stimulated genes (ISGs) were significantly upregulated. Altogether, our results provide novel evidence to support the ability of SARS-CoV-2 to infect and replicate in hESCs.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

Single-shot rAAV5-based Vaccine Provides Long-term Protective Immunity against SARS-CoV-2 and Its Variants (preprint)

The COVID-19 pandemic and the SARS-CoV-2 with its variants have posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against the SARS-CoV-2 variants. Therefore, novel vaccines to match current mutated viral lineages with long-term protective immunity are urgently in demand. In the current study, we for the first time designed a recombinant Adeno-Associated Virus 5 (rAAV5)-based vaccine named as rAAV-COVID-19 vaccine (Covacinplus) by using RBD-plus of spike protein with both the single-stranded and the self-complementary AAV5 delivering vectors (ssAAV5 and scAAAV5), which provides excellent protection from SARS-CoV-2 infection. A single dose vaccination induced the strong immune response against SARS-CoV-2. The induced neutralizing antibodies (NAs) titers were maintained at a high peak level of over 1:1024 even after more than one year of injection and accompanied with functional T-cells responses in mice. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines exhibited high levels of serum NAs against current circulating variants including variants Alpha, Beta, Gamma and Delta. SARS-CoV-2 virus challenge test showed that ssAAV5-RBD-plus vaccine protected both young and old age mice from SARS-CoV-2 infection in the upper and the lower respiratory tracts. Moreover, whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genome of the vaccinated mice after one year vaccination, demonstrating excellent safety of the vaccine. Taken together, this study suggests that rAAV5-based vaccine is powerful against SARS-CoV-2 and its variants with long-term protective immunity and excellent safety, which has great potential for development into prophylactic vaccination in human to end this global pandemic.

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery.

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.

Single-cell landscape of immunological responses in COVID-19 patients (preprint)

In COVID-19 caused by SARS-CoV-2 infection, the relationship between disease severity and the host immune response is not fully understood. Here we performed single-cell RNA sequencing in peripheral blood samples of five healthy donors and 13 COVID-19 patients including moderate, severe and convalescent cases. Through determining the transcriptional profiles of immune cells, coupled with assembled T cell receptor and B cell receptor sequences, we analyzed the functional properties of immune cells. Most cell types in COVID-19 patients showed a strong interferon-alpha response, and an overall acute inflammatory response. Moreover, intensive expansion of highly cytotoxic effector T cell subsets, such as CD4+ Effector-GNLY (Granulysin), CD8+ Effector-GNLY and NKT CD160, was associated with convalescence in moderate patients. In severe patients, the immune landscape featured a deranged interferon response, profound immune exhaustion with skewed T cell receptor repertoire and broad T cell expansion. These findings illustrate the dynamic nature of immune responses during the disease progression.

Risk Factors for In-hospital Progression of Ordinary COVID-19 in Wuhan, China: A Retrospective Cohort Study (preprint)

Objective: To describe the clinical characteristics and outcomes of ordinary COVID-19 when admitted, to describe how these patients were treated and risk factors for in-hospital progression.Methods: In this retrospective study, we included 291 adult patients diagnosed as ordinary COVID-19 on admission who had been discharged or had died between Jan 20, 2020 and Mar 16, 2020 from General Hospital of Central Theatre Command (Wuhan, China).Results: Of the 291 patients diagnosed as ordinary COVID-19 when admitted, 65 (22.34%) had been recorded COVID-19 progressing at least once, and 226 (77.66%) had been recorded COVID-19 improving during hospitalization. The median time from admission to disease progressed was 5.0 days (2.0-7.0). Multivariable regression showed increasing odds of in-hospital progression associated with male (odds ratio 2.333, 95% CI 1.135-4.395; P=0.020), preexisting cardiovascular diseases (2.433, 1.044-5.671; P=0.039), and lymphopenia (3.482, 1.783-6.799; P<0.001), elevated IL-6 (2.669, 1.084-6.574; P=0.033), d-dimer (2.829, 1.420-5.636; P=0.003) and lactate dehydrogenase (2.855, 1.458-5.591; P= 0.002) on admission.Conclusions: The potential risk factors of male, preexisting cardiovascular disease, lymphopenia, elevated IL-6, and lactate dehydrogenase, d-dimer could help clinicians to identify in-hospital progression among ordinary COVID-19 at early stage to optimize medical treatment.