Genetic Characterization of Fusion Protein of Human Respiratory Syncytial Virus: Beijing

Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further investigations in this field.The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide.Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing.Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs.Results Seventy-six samples(23.1%)were positive for HRSV.The percentage of cases in patients younger than 1year was 84.21%.Among the six Beijing isolates,four belonged to subgroup A,whose respective F genes shared97.0%-97.4%nucleotide sequence identity and 92.1%-93.0%amino acid sequence identity.The other two isolates belonged to subgroup B.Here,97.3%and 98.2%sequence identity were found at nucleotide and amino acid levels,respectively.Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other,but those two within subgroup B distributed in two distinct clusters.Subgroup A and B strains co-circulated,indicating that two different transmission chains occurred in Beijing from 2003-2004.

In vitro study on the antiviral activity of 9 extracts of traditional Chinese herbal medicine against the human respiratory syncytial virus

Objective:To study the in vitro virucidal activity of 9 extracts of traditional Chinese herbal medicine(the water extracts of Evodia lepta,Clausena lansium,Clerodendrum cyrtophyllum,Callicarpa nudiflora,Nauclea officinalis and Elaeagnus gonyanthes,the alcohol extracts of Nauclea officinalis,Elaeagnus gonyanthes and Zanthoxylumarmatum)on human respiratory syncytial virus(HRSV).Methods:The cytotoxic effect of the extracts on cells was evaluated by a cell viability assay using the CCK-8 method,a concentration of the extracts with cell viability greater than 50%was selected for the follow-up anti-HRSV effect assay,the 50%effective concentration(EC50)was assessed by an in vitro cell infection model.Results:The EC50s of the water extract from Clerodendrum cyrtophyllum,Callicarpa nudiflora and Elaeagnus gonyanthes were 0.05 mg/mL,0.03 mg/mL and 0.05 mg/mL,and the therapeutic index(TI)of them were 18.60,21.67 and 56.80 respectively.Conclusion:The water extracts of Clerodendrum cyrtophyllum,Callicarpa nudiflora and Elaeagnus gonyanthes possess the activity of anti-HRSV virus.

Surfactant Protein D for Pathological Evaluation of Infant Acute Respiratory Distress Syndrome Caused by Respiratory Syncytial Virus Infection

Pediatric respiratory syncytial viral infection (RS) usually shows relatively good outcome;however, when it accompanies acute respiratory distress syndrome (ARDS), this becomes fatal. We experienced three pediatric patients with RS + ARDS, with all showing good outcome with steroid pulse therapy. We wish to emphasize;1) steroid pulse therapy may become an option for this condition, and 2) plasma KL-6 and surfactant protein D levels may become a biomarker reflecting the disease progression/condition. Patients were, aged 1 month, 1 year 5 months, and 1 year 11 months. In all three, the respiratory condition deteriorated rapidly, requiring invasive ventilator management. Although the effectiveness of steroid treatment for ARDS is controversial, very severe condition prompted us to employ steroid pulse therapy, after which, oxygenation rapidly improved without adverse events. Plasma KL-6 and surfactant protein D levels were measured during exacerbations of ARDS, steroid pulse therapy, and recovery. Surfactant protein D levels were closely associated with oxygenation, suggesting this substance level might be a biomarker of ARDS caused by the disruption of the alveolar epithelial lining and to understand oxygenation without time lag.

Efficacy and mechanisms of Traditional Chinese Medicine prevention and treatment for respiratory viruses

Respiratory virus infection was the most common viral infection in clinical practice with the greatest impact,including the Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),posing a huge threat to the world's public health and human life safety.Commonly used antiviral drugs have obvious side effects and a narrow scope of application.Respiratory viruses are susceptible to infection,mutation,and prevalence,which also pose challenges to traditional antiviral drugs and vaccine development.Traditional Chinese Medicine(TCM)has a long history of treating infectious diseases,with many herbs and compounds.Its multi-component,multi-target and multi-path characteristics have made it have great advantages and potential in the development of new anti-respiratory virus drugs.This review summarized TCM for the prevention and treatment of common respiratory viruses,and provided new strategies for the research and development of new TCM antiviral drugs and for responding to infectious respiratory virus diseases.

Human Milk Oligosaccharides Enhance Innate Immunity to Respiratory Syncytial Virus and Influenza <i>in Vitro</i>

Human milk oligosaccharides (HMO) contribute to innate immunity by enhancing growth of beneficial bacteria, epithelial cell maturation and mucosal barrier integrity. They have immunomodulatory effects and can block pathogen binding to host cell surface glycans or receptors. We investigated the effects of 2’-fucosyllactose (2’FL), 6’-sialyllactose (6’SL), 3’-sialyllactose (3’SL) and lacto-N-neoTetraose (LNnT) on human respiratory epithelial cell lines or peripheral blood mononuclear cells (PBMCs) following respiratory viral infectionin vitro. Expression of cytokines and viral load were monitored in infected cells. These biomarkers of innate immunity were selected since viral load and cytokine levels (IP-10, MIP-1α, IL-6, IL-8, TNF-α) have been correlated with disease severity in respiratory syncytial virus (RSV) and influenza (IAV) virus infectionin vivo. 2’FL significantly decreased RSV viral load and cytokines associated with disease severity (IL-6, IL-8, MIP-1α) and inflammation (TNF-α, MCP-1) in airway epithelial cells. LNnT and 6’SL significantly decreased IAV viral load in airway epithelial cells. 6’SL dose-dependently down-regulated IP-10 and TNF-α in RSV infected PBMCs. HMO at or below levels found in breast milk enhance innate immunity to respiratory viruses in vitro and may interact directly with cells to modulate biomarkers of innate immunity.

A multi-center study on genetic variations in the fusion protein of respiratory syncytial virus from children with Acute Lower Respiratory Tract Infections in China during 2017-2021

Respiratory syncytial virus(RSV)is a significant cause of acute lower respiratory tract infection(ALRTI)in children underfive years of age.Between 2017 and 2021,396 complete sequences of the RSV F gene were obtained from 500 RSV-positive throat swabs collected from ten hospitals across nine provinces in China.In addition,151 sequences from China were sourced from GenBank and GISAID,making a total of 549 RSV F gene sequences subjected to analysis.Phylogenetic and genetic diversity analyses revealed that the RSV F genes circulating in China from 2017 to 2021 have remained relatively conserved,although some amino acids(AAs)have undergone changes.AA mutations with frequencies10%were identified at six sites and the p27 region:V384I(site I),N276S(site II),R213S(siteØ),and K124N(p27)for RSV A;F45L(site I),M152I/L172Q/S173 L/I185V/K191R(site V),and R202Q/I206M/Q209R(siteØ)for RSV B.Comparing mutational frequencies in RSV-F before and after 2020 revealed minor changes for RSV A,while the K191R,I206M,and Q209R frequencies increased by over 10%in RSV B.Notably,the nirsevimab-resistant mutation,S211N in RSV B,increased in frequency from 0%to 1.15%.Both representative strains aligned with the predicted RSV-F structures of their respective prototypes exhibited similar conformations,with low root-mean-square deviation values.These results could provide foundational data from China for the development of RSV mAbs and vaccines.

牛呼吸道合胞体病毒G和F蛋白的生物学功能

牛呼吸道合胞体病毒(bovine respiratory syncytial virus, BRSV)是一种在世界范围内引起牛呼吸道疾病的病毒性病因之一,主要引起1岁龄以下犊牛的严重下呼吸道感染。BRSV共编码9种结构蛋白,其中附着蛋白(G蛋白)和融合蛋白(F蛋白)是BRSV表面主要的包膜糖蛋白,参与病毒吸附、融合宿主细胞的过程。G和F蛋白含多种识别表位,能够刺激机体产生中和抗体反应,常常是牛呼吸道合胞体病(bovine respiratory syncytial disease, BRSD)疫苗开发所关注的重点。探究G和F蛋白在BRSV侵染中的作用机理对于病毒致病机理分析、疫苗开发具有重要意义。本文旨在对牛呼吸道合胞体病毒G和F蛋白的结构功能及有关疫苗的开发加以综述,以期为BRSV疫苗的研制和BRSD的防控提供参考。

毛细支气管炎患儿25羟基维生素D、T淋巴细胞亚群水平变化及与FeNO水平的关系

目的分析毛细支气管炎患儿25羟基维生素D[25-(OH)D3]、T淋巴细胞亚群水平变化以及与呼出气一氧化氮(FeNO)水平的关系。方法回顾性选取2019年4月至2021年7月廊坊市人民医院收治的100例毛细支气管炎患儿作为研究对象,依据患儿病情程度,将患儿分为轻度组(n=34)、中度组(n=36)和重度组(n=30),选取同期来廊坊市人民医院体检的32名健康婴幼儿作为对照组。比较4组的研究对象降钙素原、C反应蛋白(CRP)、白细胞计数、中性粒细胞计数、淋巴细胞计数、25-(OH)D3、CD3^(+)/CD4^(+)、CD3^(+)/CD56^(+)、CD3^(+)/CD8^(+)、FeNO水平。采用Pearson相关性分析FeNO与25-(OH)D3、CD3^(+)/CD4^(+)、CD3^(+)/CD8^(+)、CD3^(+)/CD56^(+)、降钙素原、淋巴细胞计数的相关性。结果不同病情毛细支气管炎患儿及对照组婴幼儿CRP、白细胞计数、中性粒细胞计数比较,差异均无统计学意义(P>0.05);重度组的降钙素原水平显著高于中度组、轻度组及对照组,差异均有统计学意义(P<0.05);重度组的淋巴细胞计数、25-(OH)D3、CD3^(+)/CD8^(+)比值、CD3^(+)/CD56^(+)比值均低于轻度组、对照组,差异均有统计学意义(P<0.05),但与中度组比较,差异无统计学意义(P>0.05);重度组、中度组的CD3^(+)/CD4^(+)水平均高于对照组、轻度组,差异均有统计学意义(P<0.05),而对照组与轻度组比较,中度组与重度组比较,差异均无统计学意义(P>0.05);重度组的FeNO水平高于中度组、轻度组、对照组,差异均有统计学意义(P<0.05),重度组与中度组比较,差异无统计学意义(P>0.05)。FeNO与25-(OH)D3、CD3^(+)/CD8^(+)、淋巴细胞计数呈负相关(P<0.05),FeNO与CD3^(+)/CD4^(+)、降钙素原呈正相关(P<0.05)。结论毛细支气管炎患儿CD3^(+)/CD4^(+)、FeNO升高,25-(OH)D3、CD3^(+)/CD8^(+)、CD3^(+)/CD56^(+)降低,且变化幅度与病情严重程度相关。FeNO与25-(OH)D3、CD3^(+)/CD8^(+)、淋巴细胞计数呈负相关,与CD3^(+)/CD4^(+)、降钙素原呈正相关。

A multi-center study on Molecular Epidemiology of Human Respiratory Syncytial Virus from Children with Acute Lower Respiratory Tract Infections in the Mainland of China between 2015 and 2019

Human respiratory syncytial virus(RSV) is a major pathogen of acute lower respiratory tract infection among young children. To investigate the prevalence and genetic characteristics of RSV in China, we performed a molecular epidemiological study during 2015–2019. A total of 964 RSV-positive specimens were identified from 5529 enrolled patients during a multi-center study. RSV subgroup A(RSV-A) was the predominant subgroup during this research period except in2016. Totally, 535 sequences of the second hypervariable region(HVR-2) of the G gene were obtained. Combined with182 Chinese sequences from GenBank, phylogenetic trees showed that 521 RSV-A sequences fell in genotypes ON1(512),NA1(6) and GA5(3), respectively;while 196 RSV-B sequences fell in BA9(193) and SAB4(3). ON1 and BA9 were the only genotypes after December 2015. Genotypes ON1 and BA9 can be separated into 10 and 7 lineages, respectively. The HVR-2 of genotype ON1 had six amino acid changes with a frequency more than 10%, while two substitutions H258 Q and H266 L were co-occurrences. The HVR-2 of genotype BA9 had nine amino acid substitutions with a frequency more than10%, while the sequences with T290 I and T312 I were all from 2018 to 2019. One N-glycosylation site at 237 was identified among ON1 sequences, while two N-glycosylation sites(296 and 310) were identified in the 60-nucleotide duplication region of BA9. To conclusion, ON1 and BA9 were the predominant genotypes in China during 2015–2019. For the genotypes ON1 and BA9, the G gene exhibited relatively high diversity and evolved continuously.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Human respiratory syncytial virus(RSV)infection is the leading cause of lower respiratory tract illness(LRTI),and no vaccine against LRTI has proven to be safe and effective in infants.Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice.The constructed recombinant plasmids harbored(5′to 3′)a T7 promoter,hammerhead ribozyme,RSV Long strain antigenomic cDNA with cold-passaged(cp)mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain(A2cpts)or further combined with SH gene deletion(A2cptsΔSH),HDV ribozyme(δ),and a T7 terminator.These vectors were subsequently co-transfected with four helper plasmids encoding N,P,L,and M2-1 viral proteins into BHK/T7-9 cells,and the recovered viruses were then passaged in Vero cells.The rescued recombinant RSVs(rRSVs)were named rRSV-Long/A2cp,rRSV-Long/A2cpts,and rRSV-Long/A2cptsΔSH,respectively,and stably passaged in vitro,without reversion to wild type(wt)at sites containing introduced mutations or deletion.Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive(ts)phenotype in vitro and in vivo,all rRSVs were significantly attenuated in vivo.Furthermore,BALB/c mice immunized with rRSVs produced Th1-biased immune response,resisted wtRSV infection,and were free from enhanced respiratory disease.We showed that the combination ofΔSH with attenuation(att)mutations of cpts contributed to improving att phenotype,efficacy,and gene stability of rRSV.By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains,we have laid an important foundation for the development of RSV live attenuated vaccines.

表达呼吸道合胞病毒融合前F蛋白的重组人5型腺病毒的构建

目的筛选并构建成功表达A亚型人呼吸道合胞病毒(Human Respiratory Syncytial Virus,HRSV)融合前构象F蛋白的重组人5型腺病毒(Recombinant Human type5 adenovirus,rHADV-5),为制备和研发HRSV重组腺病毒载体疫苗奠定基础。方法在真核表达载体pcDNA3.1(+)上插入具有不同突变位点的HRSV F蛋白编码序列,利用融合前F蛋白单克隆抗体AM14和D25筛选HRSV F蛋白突变体;通过同源重组将筛选出的前构象F蛋白突变体编码序列插入非复制型人5型腺病毒载体,在HEK293细胞中进行重组病毒的包装和扩增,经氯化铯密度梯度法获得纯化的重组病毒;应用夹心ELISA法和Western blot鉴定F蛋白的表达及构象。结果通过AM14和D25单克隆抗体筛选出了2个稳定维持在融和前构象的HRSV F蛋白序列(SC-3M和SC-5M)。将SC-3M和SC-5M编码序列插入非复制型人5型腺病毒载体,对两种重组人5型腺病毒(pAd5-SC-3M和pAd5-SC-5M)和空载体(pAd5-vector)纯化,病毒滴度分别达到2.016×10^(10),2.52×10^(10)和2.948×10^(11)IU/mL。pAd5-SC-3M和pAd5-SC-5M感染HEK293后均能高效表达HRSV融合前构象的F蛋白并维持三聚体结构,且pAd5-SC-5M感染细胞中F蛋白表达量高于pAd5-SC-3M。结论成功在体外传代细胞中获得可高效表达A型HRSV融合前F蛋白的重组人5型腺病毒,为进一步通过动物试验评价该HRSV重组腺病毒载体疫苗奠定了基础。

呼吸道合胞病毒肺炎患儿外周血单个核细胞IFITM3、IFI27表达变化及其临床意义

目的探讨呼吸道合胞病毒(RSV)肺炎患儿外周血单个核细胞干扰素诱导跨膜蛋白3(IFITM3)、干扰素诱导蛋白27(IFI27)表达变化及其临床意义。方法选择RSV肺炎患儿90例(观察组),其中病情程度低危28例、中危30例、高危32例,选择同期腹股沟斜疝患儿40例(对照组),采集所有研究对象外周静脉血,采用荧光定量PCR法检测外周血单个核细胞IFITM3、IFI27表达,采用ELISA法检测血清IL-1β、IL-6、TNF-α、IFN-γ,采用肺功能检测仪检测第1秒用力呼气容积占预计值的百分比(FEV1%pred)、FEV1/用力肺活量(FVC)。采用Pearson相关分析法分析RSV肺炎患儿外周血单个核细胞IFITM3、IFI27表达与血清IL-1β、IL-6、TNF-α、IFN-γ水平及FEV1%pred、FEV1/FVC的关系。采用受试者工作特征(ROC)曲线分析外周血单个核细胞IFITM3、IFI27表达对RSV肺炎的诊断价值。结果观察组外周血单个核细胞IFITM3、IFI27相对表达量及血清IL-1β、IL-6、TNF-α、IFN-γ水平均显著高于对照组,FEV1%pred、FEV1/FVC均显著低于对照组(P均<0.01)。随着病情程度加重,RSV肺炎患儿外周血单个核细胞IFITM3、IFI27相对表达量及血清IL-1β、IL-6、TNF-α、IFN-γ水平逐渐升高,FEV1%pred、FEV1/FVC逐渐降低(P均<0.01)。RSV肺炎患儿外周血单个核细胞IFITM3、IFI27表达与血清IL-1β、IL-6、TNF-α、IFN-γ水平均呈正相关关系,与FEV1%pred、FEV1/FVC均呈负相关关系(P均<0.05)。ROC曲线分析显示,外周血单个核细胞IFITM3、IFI27表达单独和联合预测RSV肺炎的曲线下面积(AUC)分别为0.756、0.763、0.837,外周血单个核细胞IFITM3、IFI27表达联合预测RSV肺炎的AUC高于外周血单个核细胞IFITM3、IFI27表达单独(Z分别为2.096、2.115,P均<0.05)。结论RSV肺炎患儿外周血单个核细胞IFITM3、IFI27表达显著升高,二者表达与血清IL-1β、IL-6、TNF-α、IFN-γ水平均呈正相关关系;外周血单个核细胞IFITM3、IFI27表达对RSV肺炎均有一定预测价值,二者联合预测价值更高。

更昔洛韦联合雾化重组人干扰素α1b治疗对呼吸道合胞病毒感染肺炎患儿炎症因子及免疫功能的影响

目的:分析更昔洛韦联合雾化重组人干扰素α1b治疗对呼吸道合胞病毒感染肺炎患儿炎症因子及免疫功能的影响。方法:选取本院收治的呼吸道合胞病毒感染肺炎患儿100例作为研究对象。根据随机数表法分为对照和观察两组,各50例;比较两组治疗前、后的炎性因子降钙素原(PCT)、白介素-6(IL-6)、C反应蛋白(CRP)与免疫球蛋白IgA、IgG、IgM水平,详细记录症状体征消失时间并分析治疗期间发生的不良症状。结果:治疗后两组比较,观察组持续发热、咳嗽、憋喘以及肺啰音消失时间短(P<0.05),血清PCT、IL-6与CRP水平低(P<0.05),血清IgG、IgA、IgM水平明显升高(P<0.05);治疗期间,两组恶心呕吐、白细胞降低、皮疹的总发生率比较无统计学意义(P>0.05)。结论:对呼吸道合胞病毒感染肺炎患儿实施更昔洛韦联合雾化重组人干扰素α1b联合用药可缩短症状体征消失时间,减轻机体炎症反应,提高机体免疫力,安全性相对较好。

人原代呼吸道上皮细胞培养及抗呼吸道合胞病毒活性

为建立人原代呼吸道上皮细胞(Human airway epithelial cell,hAEC)的培养方法,并在hAEC体系上探讨3-硫代吲哚类化合物RSV-A-4和免疫抑制剂代谢产物6-MMPr的抗呼吸道合胞病毒(Respiratory syncytial virus,RSV)活性及其机制,从而构建可用于RSV药物筛选及药效评价的细胞模型。采集人呼吸道上皮细胞样本,分离细胞后建立hAEC的培养方法,并进行细胞形态和活力鉴定;在hAEC体系上检测小分子化合物RSV-A-4和6-MMPr的抗RSV活性及其细胞毒性;采用实时荧光定量PCR(RT-qPCR)与Time-of-addition assay技术,探讨RSV-A-4和6-MMPr的抑制RSV复制的作用机制。培养的hAEC经鉴定:其体外培养存活率可达93.51%;RSV-A-4和6-MMPr的半数抑制浓度(Half maximal inhibitory concentration,IC_(50))分别为(207.30±4.77)μmol/L和(3191.00±6.11)μmol/L,6-MMPr的半数细胞毒性浓度(Half maximal cytotoxic concentration,CC_(50))为(95526.00±10.97)μmol/L,而RSV-A-4对hAEC未见明显细胞毒性;RSV-A-4和6-MMPr均在RSV病毒基因组复制阶段抑制RSV复制。成功建立可用于抗RSV药物筛选及体外评价的hAEC培养体系,RSV-A-4和6-MMPr在细胞水平均能有效抑制RSV复制。

布地奈德结合重组人干扰素α1b雾化吸入对RSV毛细支气管炎患儿肺功能指标的影响探讨

目的 探究对呼吸道合胞病毒(RSV)毛细支气管炎患儿应用布地奈德结合重组人干扰素α1b雾化吸入的效果及对其肺功能指标的影响。方法 选取92例RSV毛细支气管炎患儿,采用Excel表格分组法分为实验组与参照组,每组46例。参照组给予基础方案治疗,实验组给予布地奈德结合重组人干扰素α1b雾化吸入治疗。比较两组治疗效果、症状与体征消失时间、肺功能指标[潮气量(TV)、呼气峰流速(PEF)、达峰时间比(TPTEF/TE)及达峰容积比(VPTEF/VE)]及炎性因子[白细胞介素-10(IL-10)及干扰素-γ(INF-γ)]水平。结果 实验组治疗总有效率97.83%高于参照组的86.96%,组间对比差异成立(P<0.05)。实验组症状与体征消失时间中肺部啰音、三凹征、喘息、咳嗽消失时间分别为(4.65±0.88)、(2.96±0.52)、(3.70±1.08)、(5.18±1.30)d,均短于参照组的(5.41±1.13)、(4.20±0.87)、(4.22±0.96)、(2.79±1.24)d,组间对比差异成立(P<0.05)。实验组肺功能指标中TV、PEF、TPTEF/TE及VPTEF/VE分别为(7.51±1.29)ml/kg、(5.73±1.54)L/s、(26.91±7.08)%、(29.84±5.33)%,均高于参照组的(6.35±1.47)ml/kg、(4.02±0.83)L/s、(23.25±6.46)%、(26.91±4.32)%,组间对比差异成立(P<0.05)。实验组炎性因子指标中IL-10及INF-γ水平分别为(28.06±5.37)、(38.25±6.43)ng/L,均高于参照组的(15.64±7.22)、(29.36±5.24)ng/L,组间对比差异成立(P<0.05)。结论 布地奈德结合重组人干扰素α1b雾化吸入治疗RSV毛细支气管炎患儿的效果较好,临床症状与体征消失时间短,肺功能指标及炎性因子水平改善明显,具有重要临床应用价值。

An RNA polymerase I-driven human respiratory syncytial virus minigenome as a tool for quantifying virus titers and screening antiviral drug

Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.

IL2rg^(-/-)大鼠支持人RSV在体内的长期感染

目的为了克服已有人呼吸道合胞病毒(human respiratory syncytial virus,hRSV)动物模型的局限性,如半受纳性和感染持续时间短,本文利用TALEN基因编辑技术建立了IL2rg基因敲除(IL2rg^(-/-))的大鼠模型。方法用hRSV滴鼻感染该动物模型,观察感染期(0~35 d)的临床表征、体重及体温变化;记录不同时间点(滴鼻感染后第4、11、20、35天)鼻腔、气管、肺等呼吸道脏器的病毒总拷贝数;在观察终点(滴鼻感染后第35天)对感染动物的靶器官进行病理分析;观察不同时间点(滴鼻感染后第4、20、35天)外周血T、B、NK、NKT细胞的变化及不同时间点多种细胞因子的变化。结果(1)通过鼻内接种hRSV后,纯合的IL2rg基因敲除大鼠的呼吸道内能保持较高的病毒载量,鼻腔中病毒的平均峰值滴度能快速升至1×10^(10 )copies/g,至第5周时,病毒依然能维持复制,病毒载量亦可达到1×10^(7)copies/g。(2)但其鼻、气管和肺组织,无明显病变。(3)感染hRSV的IL2rg^(-/-)大鼠外周血B细胞含量有上升。(4)IL-6和MCP-1两种细胞因子都在感染前期上升,在观察终点回落。结论本研究基于TALEN技术建立了IL2rg^(-/-)大鼠模型,并发现该模型能很好地支持hRSV高水平复制和并长期感染,为抗病毒药物筛选、抗hRSV抗体体内效力评价,提供了良好的动物模型。

His-tag不影响RSV重组蛋白G1F/M2的免疫原性

目的:观察His-tag是否影响RSV重组蛋白G1F/M2的免疫原性。方法:PCR扩增G1和F/M2基因片段,插入表达载体pET-His和pET-DsbA-His中,转化E.coli BL21(DE3),IPTG诱导表达,采用Ni+螯合亲和层析法纯化得His-G1F/M2和DsbA-His-G1F/M2,将后者用凝血酶消化,再经Ni+螯合亲和层析法纯化得G1F/M2,将His-G1F/M2和G1F/M2免疫BALB/c小鼠,用ELISA测定抗体滴度,MTT法测定细胞毒性T细胞活性(CTL)。结果:两种蛋白在BALB/c小鼠中诱导的RSV特异性抗体和CTL活性无显著差异。结论:His-tag不影响RSV重组蛋白G1F/M2的免疫原性。

呼吸道合胞体病毒F蛋白二级结构分析及其B细胞表位预测

目的分析呼吸道合胞体病毒（RSV）F蛋白的二级结构并预测其B细胞表位。方法分析RSV F蛋白的二级结构,得出RSV F蛋白中可能出现的信号肽序列以及位置。基于同源建模的方法预测RSV F蛋白的三级结构,以三级结构为基础预测F蛋白中可能存在的构象B表位和线性B表位。结果 F蛋白的574个编码氨基酸中占主要比例的是丝氨酸（10.45%）、亮氨酸（10.28%）、天冬酰胺（8.71%）和苏氨酸（8.71%）;可能有7个蛋白结合位点、6个产生螺旋结构的区域;可能存在两个主要的β-折叠片蛋白;F蛋白N-末端第1-5区段为信号肽。RSV F蛋白头部主要由β-折叠片、无规则卷曲和转角组成,尾部主要由两段长的α-螺旋组成。RSV F蛋白中可能存在4个构象B表位、8个线性B表位。结论 F蛋白编码氨基酸主要为丝氨酸和亮氨酸,可能有7个蛋白结合位点、6个产生螺旋结构的区域、两个主要的β-折叠片蛋白。RSV F蛋白中可能存在4个构象B表位、8个线性B表位。

组成型表达人呼吸道合胞病毒F_(212-489)蛋白乳酸乳球菌的构建

目的构建能表达呼吸道合胞病毒截短F1蛋白(F212-489)的乳酸乳球菌,并检测表达蛋白的免疫反应性,以评估此重组菌作为口服活菌疫苗的可行性。方法 PCR扩增绿色荧光基因egfp以及截短f1基因(f212-489),经TA克隆测序后,将目的片段构建至pMG36e载体,电转化至乳酸乳球菌中鉴定并表达,通过荧光显微镜检测绿色荧光蛋白EGFP的表达,通过SDS-PAGE和Western blot检测F212-489蛋白的表达。结果荧光显微镜观察到重组菌pMG36e-egfp/MG1363有绿色荧光表达,Western blot检测到重组菌pMG36e-f212-489/MG1363中F212-489蛋白的表达。结论构建的乳酸乳球菌能够组成型表达F212-489蛋白,其蛋白具备免疫反应性。