SARS-CoV的分子生物学研究

SARS-CoV是引起严重急性呼吸道综合症(SARS)的病原体．更多地了解SARS-CoV的基因组、蛋白结构以及它与其它冠状病毒的关系,将有助于SARS疾病的防治．     

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.     

冠状病毒研究态势分析

2019年底,一种新型冠状病毒在武汉引起发热性呼吸道疾病(COVID-19),并在我国境内及周边国家持续蔓延,对人类流行病的防控提出了新的要求。基于文献计量学方法梳理了冠状病毒的研究进展,从全球视角对主要研发国家/地区、核心研究机构和热点研究方向进行了分析,旨在揭示冠状病毒的诊断、预防和治疗的研究态势,以期为当前科研联合攻关提供参考。     

SARS冠状病毒全基因组序列的变异分析

利用生物信息学软件分析不同地区来源的SARS -CoV全基因组序列的变异特征、碱基易变性及地区进化特点 ,结果表明 :SARS -CoV全基因组序列中 ,存在 4 77个变异位点 ,变异率为 0 .4 74‰。SARS -CoV碱基变异存在时间和地区特征。腺嘌呤和胸腺嘧啶相对于胞嘧啶和鸟嘌呤来说 ,更易发生变异。SARS -CoV全基因组序列的 5’端相对保守 ,而 3’端变异较活跃。动物来源的毒株与人源毒株具有极其密切联系。     

32株SARS冠状病毒基因组比对在PCR检测中的应用

进行了32株SARS冠状病毒基因组的多序列比对分析.所得无根进化树揭示SARS冠状病毒之间的同源性.同时,验证了所设计的PCR检测引物对全部32株SARS冠状病毒检测的适用性.     

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

新型冠状病毒Wuhan-Hu-1株Np空间结构及其B细胞抗原表位的预测

新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)是最新发现的一种可侵染人体的β属冠状病毒,该病毒入侵机体可引发新型冠状病毒肺炎(Coronavirus Disease 2019,COVID-19),该疫情的暴发在国内甚至国际上造成了严重...     

Molecular analysis of Coxsackievirus A24 variant isolates from three outbreaks of acute hemorrhagic conjunctivitis in 1988, 1994 and 2007 in Beijing,China

Coxsackievirus A24 variant (CVA24v) is a major pathogen that causes continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC). In China, the first confirmed outbreak of CVA24v-related AHC occurred in Beijing in 1988, followed by another two significant outbreaks respectively in 1994 and 2007, which coincides with the three-stage dynamic distribution of AHC in the world after 1970s. To illustrate the genetic characteristics of CVA24v in different periods, a total of 23 strains were isolated from those three outbreaks and the whole genome of those isolations were sequenced and analyzed. Compared with the prototype strain, the 23 strains shared four nucleotide deletions in the 5' UTR except the 0744 strain isolated in 2007. And at the 98th site, one nucleotide insertion was found in all the strains collected from 2007. From 1994 to 2007, amino acid polarity in the VP1 region at the 25th and the 32nd site were changed. Both the 3C and VP1 phylogenetic tree indicated that isolates from 1988 and 1994 belonged to Genotype III (GIII), and 2007 strains to Genotype IV (GIV). According to the Bayesian analysis based on complete genome sequence, the most recent common ancestors for the isolates in 1988, 1994 and 2007 were respectively estimated around October 1987, February 1993 and December 2004. The evolutionary rate of the CVA24v was estimated to be 7.45×10^-3 substitutions/site/year. Our study indicated that the early epidemic of CVA24v in Chinese mainland was the GIII. Point mutations and amino acid changes in different genotypes of CVA24v may generate intensity differences of the AHC outbreak. CVA24v has been evolving constantly with a relatively rapid rate.     

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PL^pro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC₅₀ value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.     

A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase

The 3C-like main peptidase 3CL(pro) is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CL(pro) and the structurally related 3C(pro) viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CL(pro) in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.     

Species Abundance in a Forest Community in South China: A Case of Poisson Lognormal Distribution

Case studies on Poisson lognormal distribution of species abundance have been rare, especially in forest communities. We propose a numerical method to fit the Poisson lognormal to the species abundance data at an evergreen mixed forest in the Dinghushan Biosphere Reserve, South China. Plants in the tree, shrub and herb layers in 25 quadrats of 20 m×20 m, 5 m×5 m, and 1 m×1 m were surveyed. Results indicated that: (i) for each layer, the observed species abundance with a similarly small median, mode, and a variance larger than the mean was reverse J-shaped and followed well the zero-truncated Poisson lognormal;(ii) the coefficient of variation, skewness and kurtosis of abundance, and two Poisson lognormal parameters (σ andμ) for shrub layer were closer to those for the herb layer than those for the tree layer; and (iii) from the tree to the shrub to the herb layer, the σ and the coefficient of variation decreased, whereas diversity increased. We suggest that: (i) the species abundance distributions in the three layers reflects the overall community characteristics; (ii) the Poisson lognormal can describe the species abundance distribution in diverse communities with a few abundant species but many rare species; and (iii) 1/σ should be an alternative measure of diversity.     

Analysis of severe human adenovirus infection outbreak in Guangdong Province,southern China in 2019

During 2018-2019, a severe human adenovirus (HAdV) infection outbreak occurred in southern China. Here, we screened 18 respiratory pathogens in 1704 children (≤ 14 years old) hospitalized with acute respiratory illness in Guangzhou, China, in 2019. In total, 151 patients had positive HAdV test results; 34.4% (52/151) of them exhibited severe illness. HAdV infection occurred throughout the year, with a peak in summer. The median patient age was 3.0 (interquartile range:1.1-5.0) years. Patients with severe HAdV infection exhibited increases in 12 clinical indexes (P ≤ 0.019) and decreases in four indexes (P ≤ 0.007), compared with patients exhibiting nonsevere infection. No significant differences were found in age or sex distribution according to HAdV infection severity (P > 0.05); however, the distributions of comorbid disease and HAdV co-infection differed according to HAdV infection severity (P < 0.05). The main epidemic types were HAdV-3 (47.0%, 71/151) and HAdV-7 (46.4%, 70/151). However, the severe illness rate was significantly higher in patients with HAdV-7 (51.4%) than in patients with HAdV-3 (19.7%) and other types of HAdV (20%) (P < 0.001). Sequencing analysis of genomes/capsid genes of 13 HAdV-7 isolates revealed high similarity to previous Chinese isolates. A representative HAdV-7 isolate exhibited a similar proliferation curve to the curve described for the epidemic HAdV-3 strain Guangzhou01 (accession no. DQ099432) (P > 0.05); the HAdV-7 isolate exhibited stronger virulence and infectivity, compared with HAdV-3 (P < 0.001). Overall, comorbid disease, HAdV co-infection, and high virulence and infectivity of HAdV-7 were critical risk factors for severe HAdV infection; these data can facilitate treatment, control, and prevention of HAdV infection.     

Functional role of a long non-coding RNA LIFR-AS1/miR-29a/TNFAIP3 axis in colorectal cancer resistance to pohotodynamic therapy

Colorectal Cancer (CRC) is one of the most common digestive system malignant tumors. Recently, PDT has been used as a first-line treatment for colon cancer; however, limited curative effect was obtained due to resistance of CRC to PDT. During the past decades, accumulating CRC-related long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs have been reported to exert diverse functions through various biological processes; their dysregulation might trigger and/or promote the pathological changes. Herein, we performed microarrays analysis to identify dysregulated lncRNAs, miRNAs and mRNAs in PDT-treated HCT116 cells to figure out the lncRNA-miRNA interactions related to the resistance of CRC to PDT treatment, and the downstream mRNA target, as well as the molecular mechanism. We found a total of 1096 lncRNAs dysregulated in PDT-treated CRC HCT116 cells; among them, LIFR-AS1 negatively interacted with miR-29a, one of the dysregulated miRNAs in PDT-treated CRC cells, to affect the resistance of CRC to PDT. LIFR-AS1 knockdown attenuated, whereas miR-29a inhibition enhanced the cellular effect of PDT on HCT116 cell proliferation and apoptosis. Furthermore, among the dysregulated mRNAs, TNFAIP3 was confirmed to be a direct target of miR-29a and exerted a similar effect to LIFR-AS1 on the cellular effects of PDT. In summary, LIFR-AS1 serves as a competitive endogenous RNA (ceRNA) for miR-29a to inhibit its expression and up-regulate downstream target TNFAIP3 expression, finally modulating the resistance of CRC to PDT. We provide an experimental basis for this lncRNA/miRNA/mRNA network being a promising target in CRC resistance to PDT treatment.     

江西省2010年柯萨奇病毒A组24型变异株的全基因组序列分析

急性出血性结膜炎(Acute hemorrhagic conjunctivitis,AHC)是目前人类最常见的眼病之一,柯萨奇病毒A组24型变异株(Coxsackievirus A24 variant,CV-A24v)是近年来报道引起该病的主要病原体。本研究选取10株来自江西省2010年AHC暴发疫情的CV-A24v,采用特异性引物扩增并测定其全基因组序列。对该10条CV-A24v的全基因组序列进行系统发育分析以及重组分析,计算本研究测定的江西10条以及GenBank中所有22条CV-A24v的全基因组序列的氨基酸置换熵值,并预测其正向选择位点。结果表明,在江西10条CV-A24v基因组序列中未检测到重组。基于全基因组序列构建的最大似然树表明江西10株CV-A24v属于GIV基因型,且分处于两条传播链。对上述32条CV-A24v序列的氨基酸置换熵值计算,共得到25个易突变位点(熵值>0.6),易突变概率最高的区段为2A区。基于Datamonkey中FUBAR和FEL模型分析,发现位于结构蛋白VP2区的234位氨基酸为两种模型共同获得的CV-A24v的正向选择位点。本研究分析了江西10株CV-A24v的全基因组序列特征,为CV-A24v引起的AHC防控工作提供了基础资料。     

PRRSV NJ-a株ORF5基因A表位的修饰与糖基化位点的突变对其DNA疫苗免疫效力的影响

为了提高PRRSVORF5基因的免疫效力,对ORF5基因进行了改造,将CpG序列和通用型辅助性T淋巴细胞表位插入A表位与B表位之间,并对N33与N51位糖基化位点进行了点突变,获得改造的ORF5基因。在此基础上构建了由两个CMV启动子调控的共表达改造的ORF5(MORF5)与ORF6基因的真核表达质粒pcDNA-M5A-6A。经Western-blot与IFA验证真核质粒的体外表达后,免疫6周龄Balb/c小鼠,利用微量中和试验检测免疫后的中和抗体,利用MTT法检测免疫后淋巴细胞的增生情况,并与未改造ORF5基因真核表达质粒pcDNA-5A-6A、弱毒疫苗以及灭活疫苗的免疫效果进行比较。结果表明,pcDNA-M5A-6A不但能够刺激免疫小鼠在较短的时间内产生更高水平的中和抗体,而且可以诱导产生更强烈的T淋巴细胞增殖反应。所构建的共表达PRRSV改造的ORF5基因与ORF6基因的DNA疫苗pcDNA-M5A-6A,能够较好的诱发小鼠产生较高的特异性针对PRRSV的中和抗体和细胞免疫应答,为研究能够更好地防制PRRSV的新型疫苗提供了新的思路。