After 15 years working in the mainstream media in China, I came to Sweden to establish my family. After almost ten years of freelancing for other media, now I created this website, www.greenpost.se hoping to spread information in sustainable development, sharing life experiences with readers both in China and Sweden, serving as a bridge.
I enjoy the good environment in Sweden, fresh air, clean water, blue sky and beautiful flowers.
陈雪霏,女,1966. 摩羯座,满族,出生在辽宁省凌海市,班吉塔镇,地藏寺村。兄弟姐妹6个当中最中间的一个。和二哥一样是全科人,就是哥姐弟妹都有,幸福感很强。万能血型,很容易打交道。喜欢高大上,但同情弱者,追求平等,公平正义,善良,是环保主义者,提倡节俭,从不浪费一粒粮食。创立瑞中桥绿色科技文化公司就是为中瑞绿色科技文化牵线搭桥。
教育程度:英国米德赛思大学可持续发展领导力硕士,英语和国际政治双学士,文学和法学学士。新西兰坎特伯雷大学访问学者。
工作经历,中国国际广播电台工作15年,驻津巴布韦两年,采访过南非总统曼德拉,津巴布韦总统穆加贝。
2008年采访过瑞典首相赖因费尔特。
2006-2010 人民网驻斯德哥尔摩特约记者。
2010-2012 新华社斯德哥尔摩报道员
2012-至今中国国际广播电台英语环球广播自由撰稿人
《环球时报》,《生命时报》特约记者。
《北欧时报》副社长,英文主编,《北欧绿色邮报网》社长,主编。
chenxuefei7@hotmail.com, wechat: chenxuefei7, facebook: chenxuefei7
The Swedish Government has presented a range of different measures to safeguard people’s lives, health and jobs in recent weeks.
“This crisis will continue for a long time. It will be tough. But our society is strong. If everyone takes their responsibility, together we will overcome it,” says Prime Minister Stefan Löfven.
On 1 February, the Government classified COVID-19 as a disease that constitutes a danger to society, opening the possibility of extraordinary communicable disease control measures.
The overall objective of the Government’s efforts is to reduce the pace of the COVID-19 virus’s spread: to ‘flatten the curve’ so that large numbers of people do not become ill at the same time.
It is important to implement the right measure at the right time, to achieve the best possible impact. The Government will take every decision necessary to safeguard people’s lives, health and jobs.
The measures taken by the Government and government agencies to reduce the pace of the virus’s spread need to be weighed against their effects on society and public health in general. The measures taken are reviewed constantly as the situation develops.
An important starting point is careful consideration of the expert knowledge contributed by government agencies. These expert agencies can make recommendations to the Government on the measures they consider should be taken, but they can also take decisions of their own.
“Our government agencies and our health care system are doing everything they can. But every person in Sweden needs to take individual responsibility. If everyone takes responsibility, we can keep the spread of the virus in check. Follow the authorities’ advice: if you have even the slightest symptoms, do not go to work and refrain from meeting other people,” says Mr Löfven.
People in Sweden have a high level of trust in government agencies. This means that a large proportion of people follow government agencies’ advice. In the current situation, people in Sweden are on the whole acting responsibly to reduce the spread of infection by, for example, restricting their social contacts.
This crisis may continue for a long time, and in order for the measures to work over time, people need to understand and accept them.
The efforts and decisions of the Government aim to:
1. Limit the spread of infection in the country
By limiting the spread of the virus, the Government aims to relieve pressure on the health care system and protect people’s lives, health and jobs.
2. Ensure that health and medical care resources are available
The Government aims to ensure that the municipalities and regions, which provide the health care, have all necessary resources. For this reason, central government will cover all costs arising as a result of the pandemic, e.g. higher costs for additional staff and protective equipment.
3. Limit the impact on critical services
To ensure that society can continue to function, the Government monitors needs and takes the decisions required to ensure that the health care, police, energy supply, communications, transport and food supply systems, for example, can maintain their activities.
4. Alleviate the impact on people and businesses
The Government has presented crisis packages to mitigate the financial impact of the pandemic on Swedish businesses, organisations and agencies, and to save people’s jobs and livings.
5. Ease concern
By continuously providing information, the Government aims to make it clear what measures are being taken, and why. The Government broadcasts important information live on its website, regeringen.se. Written information about the Government’s efforts, measures and decisions is also available there.
6. Implement the right measures at the right time
The Government is monitoring developments in the COVID-19 pandemic closely. It is taking the decisions that are needed, when they are needed, to limit the spread of the virus and counter its impact on society.
The responsibility principle
In Sweden, crisis management is built on the principle of responsibility. This means that the party responsible for a particular activity under normal circumstances is also responsible for that activity in a crisis situation.
More information and the responsible agencies
In Sweden, crisis management is built on the principle of responsibility. This means that the party responsible for an activity under normal circumstances is also responsible for it in a crisis. More information on how Sweden is governed (se below).
Complete, up-to-date and confirmed information from the responsible government agencies is available in a number of languages on krisinformation.se (see below).
“中国彩灯节”期间,喜讯频传:前不久刚移民来荷兰“工作”的星雅和武雯竟然获奖啦!不过,这奖不是颁给他们,而是它们的居住所,“中国彩灯节”荷兰策源地。周四,大熊猫全球奖(Giant Panda Global Awards)在柏林揭晓,欧维汉兹动物园(Ouwehands)里的大熊猫馆被评为“全世界第一熊猫豪宅”!该奖项是超过30万人投票一个月选出的结果!可谓是众望所归了!光入选的就有10名候选动物园。第二、第三名则花落法国的ZooParc de Beauval以及德国柏林动物园。
I know that people often say I was naive or I was an idealist because what I thought was never to put myself first, but to be fair and modest. Therefore I don’t have fear and I don’t worry too much.
Covid-19 is a virus that kills especially those who are senior and with some other diseases.
Thus we should be very careful and isolation is the best way to deal with it.
Lianhuaqingwen (LH) as traditional Chinese medicine (TCM) formula has been used to treat influenza and exerted broad-spectrum antiviral effects on a series of influenza viruses and immune regulatory effects [1]. The goal of this study is to demonstrate the antiviral activity of LH against the novel SARS-CoV-2 virus and its potential effect in regulating host immune response.
Methods
The antiviral activity of LH against SARS-CoV-2 was assessed in Vero E6 cells using CPE and plaque reduction assay. The effect of LH on virion morphology was visualized under transmission electron microscope. Pro-inflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays.
Results
LH significantly inhibited SARS-CoV-2 replication in Vero E6 cells and markedly reduced pro-inflammatory cytokines (TNF-α, IL-6, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Furthermore, LH treatment resulted in abnormal particle morphology of virion in cells.
Conclusions
LH significantly inhibits the SARS-COV-2 replication, affects virus morphology and exerts anti-inflammatory activity in vitro. These findings indicate that LH protects against the virus attack, making its use a novel strategy for controlling the COVID-19 disease.
Coronaviruses are a group of enveloped viruses named for their coronary appearance with positive single-stranded RNA genomes [2]. In addition to six known strains of coronaviruses that are infectious to humans, a novel coronavirus (SARS-CoV-2) was detected recently in Wuhan, China [3,4]. Like the other two highly pathogenic coronaviruses SARS-CoV and MERS-CoV, SARS-CoV-2 also caused severe respiratory illness and even death. Moreover, the population’s susceptibility to these highly pathogenic coronaviruses has contributed to large outbreaks and evolved into the public health events, highlighting the necessity to prepare for future reemergence or the novel emerging viruses [5].
Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 is initiated by zoonotic transmission likely from bats and spreads rapidly among humans [6]. The basic reproduction number (R0) of person-to-person spread is about at 2.6, which means that the SARS-CoV-2 infected cases grow at an exponential rate. As of February 07, 2020, 57,620 cases of the SARS-CoV-2 have been reported in China, including 26,359 suspected cases, and a sustained increase is predictable. The initial patient cluster with confirmed SARS-CoV-2 infection was reported Wuhan pneumonia with unknown aetiology, which bore some resemblance to SARS-CoV and MERS-CoV infections and was associated with ICU admission and high mortality. Moreover, High concentrations of cytokines were recorded in plasma of patients requiring ICU admission, such as GCSF, IP10, MCP1, MIP1A, and TNFα, suggesting that the cytokine storm was associated with disease severity [7]. A retrospective clinical study indicated the risk of fatality among hospitalized cases at 4.3% in single-center case series of 138 hospitalized patients [8], and the infection fatality risk could be below 1% or even below 0.1% in a large number of undetected relatively mild infections [9]. However, It is challenging to judge the severity and predict the consequences with the information available so far. Since no specific antiviral treatment for COVID-19 is currently available, supportive cares, including symptomatic controls and prevention of complications remain the most critical therapeutic regimens, especially in preventing acute respiratory distress syndrome [10]. Although the control of SARS-CoV-2 still presents multiple challenges in the short term, more potent antiviral drugs are urgent to be developed [4].
At present, some drugs are effective in eliminating SARS-CoV-2 and improving symptoms. The most promising antiviral drug for SARS-CoV-2 is remdesivir that is currently under clinical development for the treatment of Ebola virus infection [11]. However, the efficacy and safety of remdesivir for SARS-CoV-2 pneumonia patients need to be assessed by further clinical trials. In addition, in the prevention and treatment of COVID-19, Tranditonal Chinese medicines have received broad adoption, especially in treating cases of mild symptoms [12]. Lianhuaqingwen (LH), a Chinese patent medicine composed of 13 herbs, has played a positive role in the treatment of SARS-CoV-2. A retrospective analysis of clinical records was conducted in the SARS-CoV-2 infected patients at Wuhan Ninth Hospital and CR & WISCO General Hospital. LH combination could significantly relieve cardinal symptoms and reduce the course of the COVID-19 [13], making it successively included in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Fourth/Fifth/Sixth/Seventh Edition) issued by National Health Commission of the People’s Republic of China and also recommended by 20 provincial health commissions including Hubei, Beijing, and Shanghai as well as National Administration of Traditional Chinese Medicine for the treatment of COVID-19. Moreover, LH exerted broad-spectrum effects on a series of influenza viruses by inhibiting viral propagation and regulating immune function and achieved similar therapeutic effectiveness with Oseltamivir in reducing the course of H1N1 virus infection [1,14,15]. Notably, the anti-influenza activity of LH in infected mice might depend on the regulation of cytokines, particularly in cytokine storm associated cytokines, such as IP-10, MCP-1, MIP1A, and TNF-α [1]. In the present study, we evaluated the antiviral and anti-inflammatory efficiency of LH against a clinical isolate of SARS-CoV-2 from Guangzhou in vitro.
2. Materials and Methods
2.1. Cell lines and virus
The African green monkey kidney epithelial (Vero E6) cells and the human hepatocellular carcinoma (Huh-7) cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C. A clinical isolated SARS-CoV-2 virus (Genebank accession no. MT123290.1) was propagated in Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID50) according to the cytopathic effect by use of Reed-Muench method [17]. All the infection experiments were performed in a biosafety level-3 (BLS-3) laboratory.
2.2. Reagent preparation
LH capsule (Lot No.A2001108) was obtained from Yiling Pharmaceutical Co. Ltd. (Shijiazhuang, China). UPLC fingerprints of LH consist of 32 common peaks. 9 of 32 common peaks are identified. The similarities in 10 batches of LH Capsules samples were all above 0.96 (Supplementary Fig. 1). The black powder of raw material of LH was first dissolved in dimethyl sulfoxide (DMSO) to 240 mg/mL. After shaking for 30 min at room temperature, the LH solution was diluted with serum-free DMEM to 24 mg/mL as a stock solution and stored at −20 °C before using. Remdesivir was kindly provided by Prof. Jiancun Zhang from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and was dissolved in DMSO to 100 mM and stored at −20 °C before using. DMEM with 2% FBS was used as the dilution buffer in the follow-up experiments.
2.3. Cytotoxicity assay
The cytotoxic effects of the LH on Vero E6 and Huh-7 cells were evaluated by Methyl Thiazolyl Tetrazolium (MTT) assay. Briefly, monolayers of Vero E6 cells and Huh-7 cells in 96-well plates were rinsed with phosphate-buffered saline (PBS) followed by incubation with indicated concentrations of LH. After 72 h, the cells were stained with MTT solution at 0.5 mg/mL for 4 h. The supernatants were then removed, and the formed formazan crystals were dissolved in 200 μL DMSO. The absorbance was measured at 490 nm using Multiskan Spectrum reader (Thermo Fisher, USA). The 50% cytotoxic concentration (CC50) was calculated by the GraphPad Prism 7.0 software.
2.4. Cytopathic effect (CPE) inhibition assay
The Vero E6 cell monolayers were grown in 96-well plates and inoculated with 100 TCID50 of coronavirus strains at 37 ̊C for 2 h. The inoculum was removed, and the cells were subsequently incubated with indicated concentrations of LH or the positive control remdesivir. Following the 72 h of incubation, the infected cells shown 100% CPE under the microscope. The percentage of CPE in LH-treated cells were recorded. The 50% inhibition concentration (IC50) of the virus-induced CPE by LH was calculated by the Reed-Muench method [17].
2.5. Plaque reduction assay
The Vero E6 cell monolayers in 6-well plates were infected with 50 plaque-forming units (PFU) of SARS-CoV-2 for 2 h at 37 °C. After incubation, the cell monolayers were covered with agar overlay (final concentration: 0.6% agar, 2% FBS, indicated concentrations of LH or remdesivir). The plates were then incubated for 48 h at 37 °C with 5% CO2. Subsequently, the agar overlays were removed, and the cell monolayer was fixed with 10% formalin, stained with 1% crystal violet, and then the plaques were counted and photographed.
2.6. RNA isolation and reverse transcriptase-quantitative PCR analysis (RT-qPCR)
The Huh-7 cell monolayers in 12-well plate were rinsed with PBS and then exposed to coronavirus at a multiplicity of infection (MOI) of 1 for 2 h at 37 °C. The inoculum was removed and replaced with the indicated concentrations of LH or mock-treated with DMEM supplemented with 2% FBS for subsequent 48 h incubation at 37 °C with 5% CO2. The cells were then harvested for RNA isolation and qPCR as described previously [16]. The primer and probe sequences used for analysis are listed in Supplementary Table 1. The relative mRNA expression was calculated using the 2-△△Ct method with GAPDH as an internal reference gene.
2.7. Electron microscope
Monolayers of Vero E6 cells in 6-well plates were incubated with SARS-CoV-2 at a MOI of 0.001 for 2 h at 37 °C. The virus inoculum was then removed and replaced with DMEM medium supplemented with 2% FBS containing LH (600 μg/mL) or remdesivir (5 μM). At 48 h p.i., the cells were fixed, dehydrated and embedded as described previously [18]. Ultrathin sections (70 nm) of embedded cells were prepared, deposited onto Formvar-coated copper grids (200 mesh), stained with uranyl acetate and lead citrate, and then observed under JEM-1400 PLUS transmission electron microscopy (Japan Electron Optics Laboratory Co., Ltd., JEM-1400 PLUS).
2.8. Statistical Analyses
Statistical analysis was performed using GraphPad Prism 7.0 software. The differences in mRNA expression levels of cytokines were compared using a one-way analysis of variance (ANOVA). Values of p < 0.05 was considered to be statistically significant.
3. Results
3.1. Antiviral activity of LH on SARS-CoV-2 in vitro
The cell viability after LH or remdesivir treatment was determined by MTT assay in both Vero E6 and Huh-7 cells. LH showed unapparent cytotoxicity for both cell lines at concentrations up to 600 μg/mL (Fig. 1A, C). The positive control remdesivir showed no cytotoxicity to cells at a concentration of 50µM (Fig. 1B, D).
To investigate the antiviral effect of LH against SARS-CoV-2 virus, the Vero E6 cells were infected with 100 TCID50 of virus and incubated with LH at various concentrations for 72 h. As shown in Fig. 2A, LH inhibited the replication of SARS-CoV-2 virus with an IC50 value of 411.2 µg/mL by CPE assay (Fig. 2A). Meanwhile, treatment with LH following infection also had a dose-dependent inhibitory effect on plaque formation of the SARS-CoV-2 virus (Fig. 2C). We selected remdesivir as the positive control in our study and the results showed that remdesivir potently inhibited virus-induced CPE with an IC50 of 0.651 µM and a total plaque formation inhibition at 5 μM (Fig. 2B, C).
To further confirm the efficacy of LH in inhibiting SARS-CoV-2 virus replication in cells, we detected the viral particles in ultrathin sections of infected cells under electron microscopy. At 48 h p.i., viral particles were found in cytoplasm, intracellular vesicles, endoplasmic reticulum, and cell membrane and presented spherical crown-like appearance, which was typical coronavirus morphology (Fig. 3B, G). LH (600 μg/mL) and positive control remdesivir (5 μM) treatment resulted in a reduction of the number of virions compared with mock-treated infected cells (Fig. 3G–J). It was interesting to note that some virions in the surface of LH-treated cells presented spindle sharp which was in contrast to the typical spherical particles in the mock-treated cells (Fig. 3I).
3.2. Inhibition of SARS-CoV-2-induced cytokine and chemokine expression by LH in vitro
To determine the effect of LH on the expression of cytokines and chemokines induced by SAR2-CoV-2, the mRNA expression levels of TNF-α, IL-6, CCL-2/MCP-1, and CXCL-10/IP-10 were detected and compared between the LH-treated and mock-treated Huh-7 cells. The results showed that the elevated expressions of these four cytokines were significantly inhibited by LH treatment in a concentration-dependent manner (Fig. 4).
4. Discussion
Starting from December 2019, a pandemic of respiratory illness caused by a novel coronavirus named SARS-CoV-2 is sweeping the mainland of China. This virus has spread to several foreign countries, threatening to trigger a global outbreak. Several antiviral agents can be envisaged to control or prevent viral infections by antiviral assay in vitro [14,17]. However, the efficacy and safety of novel candidates need validations in vivo, even for those clinically approved medicines, which means that it will take months to years for clinical practices. At present, symptomatic and supportive treatments remain key to clinical practices. Thus, Traditional Chinese Medicines (TCM) carried both the antiviral effect and the symptomatic relief might bring more clinical benefits [12]. As a classical TCM prescription for respiratory diseases, LH is the only approved medicine in the treatment of SARS and influenza. After the outbreak of SARS-CoV-2, LH as a representative TCM prescription was recommended again in the latest Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia issued by National Health Commission of the People’s Republic of China The purpose of this study was to demonstrate whether the therapeutic effects of LH on the COVID-19 targeting virus replication and immunological regulation as it did on the infection caused by influenza viruses.
Our previous study showed that LH exhibited in vitro anti-influenza activity with IC50 ranging from 200-2000 μg/mL [1]. Here we demonstrated that LH also has a comparable antiviral potency against the SARS-CoV-2 virus with an IC50 value of 411.2 μg/mL (Fig. 2). Transmission electron microscopy (TEM) has been a potent tool to observe virus entry, virus particle assembly, viral ultrastructure, and budding from the plasma membrane [17]. To understand the antiviral details of LH, EM pictures were taken from each group. Abundant virus particles assembled at the surface of membrane, cytoplasm, and plasma vesicles in the SARS-CoV-2 infected cells, decreased in the treatment of LH at 600ug/mL. Notably, slight deformation of virus particles was seen in the LH treatment, which required us to make further studies.
Highly pathogenic coronaviruses such as SARS-CoV and MERS-CoV cause fatal pneumonia, which is mainly associated with rapid virus replication, massive inflammatory cell infiltration and elevated proinflammatory cytokine/chemokine responses. Although the pathophysiology of fatal pneumonia caused by highly pathogenic coronaviruses has not been completely understood, accumulating evidence suggests that the cytokine storm plays a crucial role in causing fatal pneumonia [18]. Excessive amounts of proinflammatory cytokines were reported (e.g., IL-1β, IL-6, IL-12, IFN-γ, IP-10, and MCP-1) in the serum of SARS patients [18], similar in the serum of MERS patients [19]. Chaolin Huang et al. confirmed the occurrence of the cytokine storm in the COVID-19 patients in ICU rather than those in non-ICU patients [7]. Based on the excessive cytokines responses, Suxin Wan et al. claimed that IL-6 and IL-10 levels could be used as one of the bases for predicting the outcome and prognosis of the COVID-2019 [20]. In this study, host cells infected with HCoV-229E and SARS-COV-2 increased the cytokine release such as TNF-α, IL-6, CCL-2/MCP-1, and CXCL-10/IP-10, which was suppressed by LH in a dose-dependent manner. The change of cytokine profiles suggested that LH might have a potential effect on the inhibition of cytokine storm induced by SARS-COV-2, which also needed to be validated in vivo.
5. Conclusion
Since the launch of LH, it has been widely used as a broad spectrum of antiviral agent in the clinical practice, especially for various respiratory virus infections. Previous studies have shown that LH a broad spectrum of effects on a series of influenza viruses by interfering with both viral and host reactions. Although LH significantly relieved the clinical symptoms of the COVID-19, the underlying mechanism of antiviral effects on coronavirus, especially in the SARS-COV-2, was still elusive. In this study, we demonstrated that LH exerted its anti-coronavirus activity by inhibiting virus replication and reducing the cytokine release from host cells, which supported the clinical application of LH in combination with existing therapies to treat COVID-2019.
Acknowledgements
The study was funded by Beijing Municipal Science and Technology Commission NCP Emergency Project; Hebei Provincial Department of Science and Technology NCP prevention and control emergency scientific research project (Grant no. 20277708D); The Science research project of the Guangdong Province (Grant no. 2020B111110001); Daxing District Science and technology development projects (Grant no. KT202008013).
Appendix A. Supplementary data
Download all supplementary files included with this articleHelp
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