Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses (preprint)

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically-determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus SARS-CoV-2, resolved at 4.7 [A] resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the Ts "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4-6.9 [A] resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across the studied human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9-8.0 [A] resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4-9.0 [A] resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities, with implications for potential protein-binding modes and therapeutic targets. SignificanceThe three-dimensional structures of viral RNAs are of interest to the study of viral pathogenesis and therapeutic design, but the three-dimensional structures of viral RNAs remain poorly characterized. Here, we provide the first 3D structures of the SL5 domain (124-160 nt, 40.0-51.4 kDa) from the majority of human-infecting coronaviruses. All studied SL5s exhibit a similar 4-way junction, with their crossing angles grouped along phylogenetic boundaries. Further, across all species studied, conserved UUYYGU hexaloop pairs are located at opposing ends of a coaxial stack, suggesting that their three-dimensional arrangement is important for their as-of-yet defined function. These conserved tertiary features support the relevance of SL5 for pan-coronavirus fitness and highlight new routes in understanding its molecular and virological roles and in developing SL5-based antivirals. Classification: Biological Sciences, Biophysics and Computational Biology

Potential molecular mechanism of Compound Yizhihao Granules in treatment of COVID-19 based on network pharmacology and molecular docking method

Objective: To explore the potential molecular mechanism of Compound Yizhihao Granules in treatment of coronavirus disease 2019 (COVID-19) through network pharmacology and molecular docking technology.

Optimal metabolic states in cells (preprint)

Cells, in order to thrive, make efficient use of metabolites, proteins, energy, membrane space, and time. How, for example, should they allocate the available amount of protein to different metabolic pathways or cell functions? To model metabolic behaviour as an economic problem, some flux analysis model, kinetic models, and cell models apply optimality principles. However, due to their different assumptions these models are hard to compare and combine. Benefits and costs of metabolic pathways - e.g. favouring high production fluxes and low metabolite and enzyme cost - can be derived from general fitness objectives such as fast cell growth. To define pathway objectives, we may assume "optimistically" that, given a pathway state, any cell variables outside the pathway will be chosen for maximal fitness. The resulting fitness defines an effective pathway objective as a function of the pathway variables. Here I propose a unified theory that considers kinetic models, describes the set of feasible states as a state manifold and score each state by cost and benefit functions for fluxes, metabolite concentrations, and enzyme levels. To screen the state manifold and to find optimal states, the problem can be projected into flux, metabolite, or enzyme space, where effective cost and benefit functions are used. We reobtain existing modelling approaches such as enzyme cost minimisation or nonlinear versions of Flux Balance Analysis. Due to their common origin, the different approaches share mathematical optimality conditions of the same form. A general theory of optimal metabolic states, as proposed here, provides a logical link between existing modelling approaches and can help justify, interconvert, and combine metabolic optimality problems.

Assessing the Impacts of Meteorological Factors on COVID-19 Pandemic Using Generalized Estimating Equations

Background Meteorological factors have been proven to affect pathogens;both the transmission routes and other intermediate. Many studies have worked on assessing how those meteorological factors would influence the transmissibility of COVID-19. In this study, we used generalized estimating equations to evaluate the impact of meteorological factors on Coronavirus disease 2019 (COVID-19) by using three outcome variables, which are transmissibility, incidence rate, and the number of reported cases. Methods In this study, the data on the daily number of new cases and deaths of COVID-19 in 30 provinces and cities nationwide were obtained from the provincial and municipal health committees, while the data from 682 conventional weather stations in the selected provinces and cities were obtained from the website of the China Meteorological Administration. We built a Susceptible-Exposed-Symptomatic-Asymptomatic-Recovered/Removed (SEIAR) model to fit the data, then we calculated the transmissibility of COVID-19 using an indicator of the effective reproduction number (Reff). To quantify the different impacts of meteorological factors on several outcome variables including transmissibility, incidence rate, and the number of reported cases of COVID-19, we collected panel data and used generalized estimating equations. We also explored whether there is a lag effect and the different times of meteorological factors on the three outcome variables. Results Precipitation and wind speed had a negative effect on transmissibility, incidence rate, and the number of reported cases, while humidity had a positive effect on them. The higher the temperature, the lower the transmissibility. The temperature had a lag effect on the incidence rate, while the remaining five meteorological factors had immediate and lag effects on the incidence rate and the number of reported cases. Conclusion Meteorological factors had similar effects on incidence rate and number of reported cases, but different effects on transmissibility. Temperature, relative humidity, precipitation, sunshine hours, and wind speed had immediate and lag effects on transmissibility, but with different lag times. An increase in temperature may first cause a decrease in virus transmissibility and then lead to a decrease in incidence rate. Also, the mechanism of the role of meteorological factors in the process of transmissibility to incidence rate needs to be further explored.

COVID-19 and A-share banks' stock price volatility: From the perspective of the epidemic evolution in China and the US

With a financial market dominated by indirect financing, China's banking system played a critical role in the government's response to COVID-19, which piqued our interest in the short-term impact of COVID-19 on the risk of China's banks. Examining the stock price of A-share listed banks and the number of confirmed cases in China and the US during the short time window surrounding the COVID-19 pandemic's outbreak, this study reveals that COVID-19 increased the A-share banking price volatility in both China and the US, reflecting a strong spillover effect of the US economic and financial system. Furthermore, COVID-19 in China has a smaller impact on the stock price volatility of China's state-owned banks (SOBs) than that of medium- and small-sized (M&S) banks, reflecting the higher risk resistance capability of large SOBs. Further analysis confirms that the impact primarily reflected systematic risk rather than idiosyncratic risk, as small and micro enterprises and M&S banks received more targeted financial support from the government. In contrast, large banks took on more responsibilities in the emergency financial stimulus, narrowing the idiosyncratic risk gap between the two types of banks and allowing the banking industry to better play its core role in the recovery of real economy in China. These findings will assist us in better understanding the effectiveness of financial assistance policies during the epidemic and will provide insights for future policymaking during similar crises.

Accelerating PERx Reaction Enables Covalent Nanobodies for Potent Neutralization of SARS-Cov-2 and Variants (preprint)

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here we developed covalent nanobodies capable of binding with SARS-CoV-2 spike protein irreversibly via proximity-enabled reactive therapeutic (PERx) mechanism. A novel latent bioreactive amino acid FFY was designed and genetically encoded into nanobodies to accelerate PERx reaction rate. After covalent engineering, nanobodies binding with the Spike in the down state, but not in the up state, were discovered to possess striking enhancement in inhibiting viral infection. In comparison with the noncovalent wildtype nanobody, the FFY-incorporated covalent nanobody neutralized both authentic SARS-CoV-2 and its Alpha and Delta variants with potency drastically increased over tens of folds. This PERx-enabled covalent nanobody strategy and uncovered insights on potency increase can be valuable to developing effective therapeutics for various viral infections.

Long-term Improvement of Air Quality Associated with Lung Function Benefits in Chinese Young Adults: A Quasi-experiment Cohort Study (preprint)

Background: Long-term exposure to air pollution is associated with lung function impairment. However, whether long-term improvements in air quality could improve lung function is unclear. Methods: : We conducted a prospective quasi-experiment cohort study with 1731 college students in Shandong, China from September 2019 to September 2020, covering COVID-19 lockdown period. Data on PM 2.5 , PM 10 , NO 2 and SO 2 concentrations were obtained from China Environmental Monitoring Station. The concentration of O3 was obtained from Tracking Air Pollution in China. Lung function indicators included forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1) and forced expiratory flow at 50% of FVC (FEF50%). Linear mixed-effects model was used to examine the associations between the change of air pollutants’ concentrations and the change of lung functions. We also conducted stratified analysis by sex. Results: : Compared with 2019, the mean FVC, FEV1 and FEF50% were elevated by 414.4ml, 321.5ml, and 28.4ml respectively in 2020. Every 5μg/m 3 decrease in annual average PM 2.5 concentrations was associated with 36.0ml [95% confidence interval (CI):6.0, 66.0ml], 46.1ml (95% CI:16.7, 75.5ml), and 124.2ml/s (95% CI:69.5, 178.9ml/s) increment in the FVC, FEV1, and FEF50%, respectively. Similar associations were found for PM 10 . There was no significant effect difference between male and female. Conclusions: : Long-term improvement of air quality can improve lung function among young adults. Stricter policies on improving air quality are needed to protect human health. Funding Taishan Scholar Program

The Association between Cold Temperature and Mortality During and Before the COVID-19 Pandemic in Italy: A Nationwide Time-Stratified Case-Crossover Study (preprint)

Background: The coronavirus disease 2019 (COVID-19) pandemic and related containment measures have changed human lives and behaviours. Whether the pandemic could change the association between cold temperature and mortality remains unknown. We aimed to assess whether the association between cold temperature and all-cause mortality in the pandemic period has changed or not compared to the non-COVID-19 period (2015-2019) in Italy.Methods: We collected daily all-cause mortality data and meteorological data for 107 Italian provinces from 1, January 2015 to 31, May 2020. A time-stratified case-crossover design with the distributed lag non-linear model was used to examine the association between cold temperature and all-cause mortality during the first three months (from March to May in 2020) of the COVID-19 outbreak and the same months in 2015-2019.Findings: The relative risk (RR) of all-cause mortality at extreme cold temperature (4 ˚C) in comparison with the minimum mortality temperature (24 ˚C) was 4·75 [95% confidence interval (CI): 3·90-5·79] in the pandemic period, which is more than triple higher than RR [1·41 (95%CI: 1·33-1·50)] in the same months during 2015-2019. The shift in cold-mortality association was particularly significant for people aged 65-74 years [RR (95%CI): 5·98 (3·78-9·46) in 2020 versus 1·29 (1·10-1·51) in 2015-2019], 75-84 years [5·25 (3·79-7·26) versus 1·40 (1·25-1·56)], and ≥ 85 years [5·03 (3·90-6·51) versus 1·52 (1·39-1·66)], but not significant for those aged 0-64 years [1·95 (1·17-3·24) versus 1·24 (1·05-1·48)].Interpretation: The findings suggest that the COVID-19 pandemic enhanced the risk of cold temperature on mortality in Italy, particularly among the elderly people. Further studies are warranted to understand the exact mechanism when detailed data are available.Funding: YG was supported by a Career Development Fellowship of the Australian National Health and Medical Research Council (#APP1107107 & #APP1163693). SL was supported by an Early Career Fellowship of Australian National Health and Medical Research Council (#APP1109193). RX and TY were supported by China Scholarship Council (201806010405, 201906320051). Declaration of Interests: None.Ethics Approval Statement: This study was approved by the School of Public Health and Preventive Medicine, Monash University.

Vulnerability and burden of all-cause mortality associated with particulate air pollution increased during COVID-19 pandemic: a nationwide observed study in Italy (preprint)

Abstract Background: Limited evidence is available on the health effects of particulate matter (i.e. PM2.5, particulate matter with an aerodynamic diameter < 2.5m; PM10, < 10m; PM2.5-10, 2.5-10m) during the pandemic of COVID-19 in Italy. Objectives: To examine the associations between all-cause mortality and daily PM2.5, PM2.5-10, and PM10 in the pandemic period, and compare them to the normal periods (2015-2019) in Italy. Methods: We collected daily data regarding all-cause (stratified by age and gender), and PM2.5, PM2.5-10, and PM10 for 107 Italian provinces from 1, January 2015 to 31, May 2020. A time-stratified case-cross design with the distributed lag non-linear model was used to examine the association between PM and all-cause mortality during the first three months of the COVID-19 outbreak (March to May in 2020) and the same months in 2015-2019. We also compared the counts and fractions of death attributable to PM in two periods. Results: Overall, Italy saw an increase in daily death counts while slight decreases in PM concentrations in 2020 pandemic period compared to same months of 2015-2019. Mortality effects were significant in lag 0-3 days for PM2.5, lag 0-2 for PM10, and lag 0-1 for PM2.5-10. Each 10 g/m3 increase in PM was associated much higher increase in daily all-cause mortality during 2020 pandemic period compared to the same months during 2015-2019 [increased mortality rate: 7.24 % (95%CI: 4.84%, 9.70%) versus 1.69% (95%CI: 1.12%, 2.25%) for PM2.5; 3.45 % (95%C: 2.58%, 4.34%) versus 1.11% (95%CI: 0.79%, 1.42%) for PM10, 4.25% (95%CI: 2.99%, 5.52%) versus 1.76% (95%CI: 1.14%, 2.38%) for PM2.5-10]. The counts and fractions of deaths attributable to PM were higher in 2020 than the normal periods for PM2.5 (attributable death counts: 20,062 in 2020 versus 3,927 per year in 2015-2019; attributable fractions: 10.2% versus 2.4%), PM10 (15,112 versus 3,999; 7.7% versus 2.5%), and PM2.5-10 (7,193 versus 2303; 3.7% versus 1.4%). Conclusions: COVID-19 pandemic increased the vulnerability and excess cases of all-cause mortality associated with short-term exposure to PM2.5, PM2.5-10 and PM10 in Italy, despite a decline in air pollution level. This suggests using historical PM-mortality association to calculate health benefits associated with reduction in PMs has big uncertainties.

COVID-19 pandemic increased the magnitude of mortality risks associated with cold temperature in Italy: A nationwide time-stratified case-crossover study (preprint)

Abstract Backgrounds: The coronavirus disease 2019 (COVID-19) pandemic and some containment measures have changed many people lives and behaviours. Whether the pandemic could change the association between cold temperature and mortality remains unknown. Objectives: We aimed to assess whether the association between cold temperature and all-cause mortality in the pandemic period has changed compared to non-COVID-19 period (2015-2019) in Italy. Methods: We collected daily all-cause mortality data and meteorological data for 107 Italian provinces from 1, January 2015 to 31, May 2020. A time-stratified case-crossover design with the distributed lag non-linear model was used to examine the association between cold temperature and all-cause mortality during the first three months (from March to May in 2020) of the COVID-19 outbreak and the same months in 2015-2019. Results: The relative risk (RR) of all-cause mortality at extreme cold temperature (2.5th percentile of temperature at 3 {degrees}C) in comparison with the minimum mortality temperature (24 {degrees}C) was 4.75 [95% confidence interval (CI): 3.90-5.79] in the pandemic period, which is more than triple higher than RR [1.41 (95%CI: 1.33-1.50)] in the same months during 2015-2019. The shift in cold-mortality association was particularly significant for people aged 65-74 years [RR (95%CI): 5.98 (3.78-9.46) in 2020 versus 1.29 (1.10-1.51) in 2015-2019], 75-84 years [5.25 (3.79-7.26) versus 1.40 (1.25-1.56)], and [≥] 85 years [5.03 (3.90-6.51) versus 1.52 (1.39-1.66)], but not significant for those aged 0-64 years [1.95 (1.17-3.24) versus 1.24 (1.05-1.48)]. Conclusion: The findings suggest that the COVID-19 pandemic enhanced the risk of cold temperature on mortality in Italy, particularly among the elderly people. Further studies are warranted to understand the exact mechanism when detailed data are available.

A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice (preprint)

Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (S{Delta}C-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of S{Delta}C-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with S{Delta}C-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

A PCR amplicon-based SARS-CoV-2 replicon for antiviral screening (preprint)

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral screening; however, SARS-CoV-2 replicon has not been reported yet. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3,000 times higher luminescence than MOCK control cells at 24 hours post-electroporation, indicating robust viral translation and RNA replication. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 M, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 M). Taken together, this system could be applied to the safe and effective antiviral screening without using infectious SARS-CoV-2. Because this is a transient replicon, further improvement including the establishment of stable cell line must be achieved.

A 3.4-Å cryo-EM structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles (preprint)

Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV and SARS-CoV-2. Here we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-Å resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a 3-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other amino acid positions as well as differences in glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies, or drugs, in the native state of the coronavirus particles.

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome (preprint)

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.