Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection.

More than 100 arboviruses, almost all of which have an RNA genome, cause disease in humans. RNA viruses are causing unprecedented health system challenges worldwide, many with little or no specific therapies or vaccines available. Certain species of mosquito can carry dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), where co-infection of these viruses has occurred. Here, we found that purified synthetic defective interfering particles (DIPs) derived from DENV type 2 (DENV-2) strongly suppressed replication of the aforementioned viruses, respiratory syncytial virus (RSV) and also the novel emerging virus SARS-CoV-2 in human cells. DENV DIPs produced in bioreactors, purified by column chromatography, and concentrated are virus-like particles that are about half the diameter of a typical DENV particle, but with similar ratios of the viral structural proteins envelope and capsid. Overall, DIP-treated cells inhibited DENV, ZIKV, YFV, RSV, and SARS-CoV-2 by at least 98% by mechanisms which included interferon (IFN)-dependent cellular antiviral responses. IMPORTANCE DIPs are spontaneously derived virus mutants with deletions in genes that block viral replication. DIPs play important roles in modulation of viral disease, innate immune responses, virus persistence and virus evolution. Here, we investigated the antiviral activity of highly purified synthetic DIPs derived from DENV, which were produced in bioreactors. DENV DIPs purified by column chromatography strongly inhibited five different RNA viruses, including DENV, ZIKV, YFV, RSV, and SARS-CoV-2 in human cells. DENV DIPs inhibited virus replication via delivery of a small, noninfectious viral RNA that activated cellular innate immunity, resulting in robust type 1 interferon responses. The work here presents a pathway for DIP production which is adaptable to Good Manufacturing Practice, so that their preclinical testing should be suitable for evaluation in subjects.

Ginkgolic acid and anacardic acid are reversible inhibitors of SARS-CoV-2 3-chymotrypsin-like protease.

Because of the emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different regions of the world, the battle with infectious coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been seesawing. Therefore, the identification of antiviral drugs is of particular importance. In order to rapidly identify inhibitors for SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), an enzyme essential for viral replication, we combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) and developed a novel sandwich-like FP screening assay. Through high-throughput screening, two hits of 3CLpro inhibitors, ginkgolic acid (GA) and anacardic acid (AA) were identified, which showed IC50 values of 11.29 ± 0.48 and 12.19 ± 0.50 µM, respectively. Their binding modes were evaluated by HPLC-Q-TOF-MS. There was no mass increase detected for SARS-CoV-2 3CLpro incubated with either GA or AA, indicating the absence of covalent adducts. The kinetic analysis clearly demonstrated that both GA and AA inhibit SARS-CoV-2 3CLpro via reversible and mixed-inhibition manner. Our results argue against conclusion that GA and AA act as irreversible and covalent inhibitors against SARS-CoV-2 3CLpro, which is based on the studies by Chen et al.

[Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay].

For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 µmol/L Mpro and 5 µmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1, 4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.

Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. RESULTS: In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. CONCLUSIONS: This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity.

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.

Predicting the effect of confinement on the COVID-19 spread using machine learning enriched with satellite air pollution observations.

The real-time monitoring of reductions of economic activity by containment measures and its effect on the transmission of the coronavirus (COVID-19) is a critical unanswered question. We inferred 5,642 weekly activity anomalies from the meteorology-adjusted differences in spaceborne tropospheric NO2 column concentrations after the 2020 COVID-19 outbreak relative to the baseline from 2016 to 2019. Two satellite observations reveal reincreasing economic activity associated with lifting control measures that comes together with accelerating COVID-19 cases before the winter of 2020/2021. Application of the near-real-time satellite NO2 observations produces a much better prediction of the deceleration of COVID-19 cases than applying the Oxford Government Response Tracker, the Public Health and Social Measures, or human mobility data as alternative predictors. A convergent cross-mapping suggests that economic activity reduction inferred from NO2 is a driver of case deceleration in most of the territories. This effect, however, is not linear, while further activity reductions were associated with weaker deceleration. Over the winter of 2020/2021, nearly 1 million daily COVID-19 cases could have been avoided by optimizing the timing and strength of activity reduction relative to a scenario based on the real distribution. Our study shows how satellite observations can provide surrogate data for activity reduction during the COVID-19 pandemic and monitor the effectiveness of containment to the pandemic before vaccines become widely available.

Multiple organ injury on admission predicts in-hospital mortality in patients with COVID-19.

Multiorgan injury has been implicated in patients with coronavirus disease 2019 (COVID-19). We aim to assess the impact of organ injury (OI) on prognosis according to the number of affected organs at admission. This is a retrospective cohort study of patients with confirmed COVID-19 in Wuhan Third Hospital & Tongren Hospital of Wuhan University from February 17 to March 22, 2020. We classified the patients according to the presence and number of damaged organs (heart, liver, and kidney). The percentage of patients with no, one, two, or three organs affected was 59.75%, 30.46%, 8.07%, and 1.72%, respectively. With the increasing number of OI, there is a tendency of gradual increase regarding the white blood cell counts, neutrophil counts, levels of C-reactive protein (CRP), lactate dehydrogenase, D-dimer, and fibrinogen as well as the incidence of most complications. In a Cox regression model, individuals with OI, old age, and an abnormal level of CRP were at a higher risk of death compared with those without. Patients with three organ injuries had the highest mortality rate (57.9%; hazard ratio [HR] with 95% confidence interval [CI] vs. patients without OI: 22.31 [10.42-47.77], those with two [23.6%; HR = 8.68, 95% CI = 4.58-16.48], one [8.6%; HR = 3.1, 95% CI = 1.7-5.7], or no OI [2.6%]; p < .001). The increasing number of OI was associated with a high risk of mortality in COVID-19 infection.

The unique features of SARS-CoV-2 transmission: Comparison with SARS-CoV, MERS-CoV and 2009 H1N1 pandemic influenza virus.

From 2002 to 2019, three deadly human coronaviruses (hCoVs), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged to produce outbreaks of SARS, MERS and coronavirus disease 2019 (Covid-19), respectively. All three hCoVs are members of the Betacoronavirus genus in the subfamily Orthocoronavirinae and share many similarities in virology and epidemiology. However, the pattern and scale of Covid-19 global spread is similar to 2009 pandemic H1N1 influenza (H1N1pdm09), rather than SARS or MERS. Covid-19 exhibits high viral shedding in the upper respiratory tract at an early stage of infection, and has a high proportion of transmission competent individuals that are pre-symptomatic, asymptomatic and mildly symptomatic, characteristics seen in H1N1pdm09 but not in SARS or MERS. These two traits of Covid-19 and H1N1pdm09 result in reduced efficiency in identification of transmission sources by symptomatic screening and play important roles in their ability to spread unchecked to cause pandemics. To overcome these attributes of Covid-19 in community transmission, identifying the transmission source by testing for virus shedding and interrupting chains of transmission by social distancing and public masking are required.

Neutrophil to lymphocyte ratio as prognostic and predictive factor in patients with coronavirus disease 2019: A retrospective cross-sectional study.

This retrospective study was designed to explore whether neutrophil to lymphocyte ratio (NLR) is a prognostic factor in patients with coronavirus disease 2019 (COVID-19). A cohort of patients with COVID-19 admitted to the Tongren Hospital of Wuhan University from 11 January 2020 to 3 March 2020 was retrospectively analyzed. Patients with hematologic malignancy were excluded. The NLR was calculated by dividing the neutrophil count by the lymphocyte count. NLR values were measured at the time of admission. The primary outcome was all-cause in-hospital mortality. A multivariate logistic analysis was performed. A total of 1004 patients with COVID-19 were included in this study. The mortality rate was 4.0% (40 cases). The median age of nonsurvivors (68 years) was significantly older than survivors (62 years). Male sex was more predominant in nonsurvival group (27; 67.5%) than in the survival group (466; 48.3%). NLR value of nonsurvival group (median: 49.06; interquartile range [IQR]: 25.71-69.70) was higher than that of survival group (median: 4.11; IQR: 2.44-8.12; P < .001). In multivariate logistic regression analysis, after adjusting for confounding factors, NLR more than 11.75 was significantly correlated with all-cause in-hospital mortality (odds ratio = 44.351; 95% confidence interval = 4.627-425.088). These results suggest that the NLR at hospital admission is associated with in-hospital mortality among patients with COVID-19. Therefore, the NLR appears to be a significant prognostic biomarker of outcomes in critically ill patients with COVID-19. However, further investigation is needed to validate this relationship with data collected prospectively.