噬菌体展示技术筛选甲型H1N1流感病毒抗原模拟表位的研究

目的筛选特异性甲型H1N1流感病毒抗原模拟表位。方法应用噬菌体表面展示技术,以2009年甲型H1N1流感康复患者的血清作为固相筛选分子,对人工合成的噬菌体随机环七肽库进行3轮吸附-洗脱-扩增筛选。从顶部琼脂噬菌斑中随机挑取32个克隆至处于对数生长期的大肠埃希菌中培养,采用ELISA,交叉反应以及竞争抑制性实验筛选并鉴定阳性克隆,对阳性克隆进行DNA序列分析,对其编码的展示于噬菌体表面的氨基酸序列与流感病毒血凝素(HA)基因进行同源性比较,确定甲型H1N1流感病毒抗原的模拟表位。结果从随机挑选的32个克隆中得到12个阳性克隆,确定氨基酸序列PLHARLP为甲型H1N1流感病毒抗原的模拟表位,其表位由HA的第52,53,59,60,61,83,271位氨基酸共同构成。结论用噬菌体环七肽库筛选得到甲型H1N1流感病毒的模拟表位,为开展用流感病毒模拟表位探索新的流感防治方法奠定了基础。     

新甲型H1N1流感病毒分子生物学研究进展

甲型流感是由甲型流感病毒引起,可致人或其他动物宿主发生不同程度呼吸道症状的疾病。2009年3月新甲型H1N1流感突然在墨西哥暴发,由于疫情迅速扩散,世界卫生组织（WHO）于2009～06—11将流感流行级别调整为最高级六级,即宣布新甲型H1N1流感发生全球流行。现病毒已传播至212个国家和地区,并处于继续扩散的状态,由于感染人数巨大,目前世卫组织已无法统计罹患人数,仅统计报告确诊死亡人数（至2010—02—19死亡15921人）。     

埃博拉病毒NP抗原表位区段的克隆和表达

目的分析埃博拉病毒核蛋白（ NP）抗原表位,克隆表达埃博拉病毒NP抗原,为免疫学诊断试剂的研究奠定基础。方法采用BioSun生物学软件预测分析埃博拉病毒NP抗原的氨基酸表位区间,采用大肠杆菌优势密码子反向翻译成基因序列,采用PCR退火合成法合成NP优势密码子抗原基因,并利用载体pBVIL1进行克隆表达。采用间接ELISA技术评价获得NP抗原的特异性。结果确定埃博拉病毒NP抗原的氨基酸表位位于360～739 aa,共设计36条引物,扩增合成1140 bp的NP抗原基因,克隆表达后,融合蛋白相对分子质量为58×103,测序结果显示插入的NP基因正确。初步结果显示,NP抗原的特异性为99．24％（130／131）。结论获得埃博拉病毒NP抗原,为进一步研制特异的埃博拉病毒快速诊断试剂提供了抗原储备。     

大流行甲型H1N1流感病毒变异株的小鼠感染实验研究

目的对甲型H1N1流感病毒变异株的小鼠感染特征进行观察。方法经过体外鸡胚/MDCK细胞传代和筛选,获得1株甲型H1N1流感病毒变异株（vCA07）;使用不同剂量流感病毒滴鼻感染BALB/c小鼠后,计算半数致死量和半数感染量,观察小鼠感染后的体质量变化、肺部病毒增殖、肺部组织病理变化等。结果与野生型毒株相比,甲型H1N1流感病毒变异株对BALB/c小鼠的致病性显著提高,其可在小鼠肺组织中高效增殖,并导致显著的病理损伤。结论甲型H1N1流感病毒变异株感染特征得以明确,为进一步建立高致病性甲型H1N1流感病毒的小鼠感染模型以及大流行甲型H1N1流感病毒致病机制的研究奠定了基础。     

甲型H1N1流感病毒隐性感染者1例

甲型H1N1流感自今年3月暴发以来,虽然各国政府始终在积极采取各种防控措施,但疫情仍在不断蔓延和扩大.WHO报告,截至2009年7月5日,此起流感疫情已波及全球五大洲的126个国家和地区,确诊病例达89 922例,死亡382人.我周内地也有24个省份累计报告确诊病例1 040例,包括输入性病例758例,本土病例282例.     

2009甲型H1N1亚型流感病毒抗原性及遗传特性研究

当人群体缺乏对某种流感病毒的免疫力或免疫力低下,而该病毒在人群中存在且能在人与人之间有效传播时,极易引起全球流感大暴发.历史上引起3次流感大暴发的流感病毒(1918年H1N1、1957年H2N2和1968年H3N2)(H或HA:hemagglutinin,血凝素;N或NA:neuraminidase,神经氨酸酶)全部或部分基因组均来源于动物流感病毒,特别是病毒HA基因最终均可追溯到禽流感病毒.     

甲型H1N1流感病毒性肺炎的影像学表现

目的:回顾性讨论12例甲型H1N1流感病毒性肺炎患者的胸部X线和CT表现特征。方法:经临床证实的甲型H1N1流感病毒性肺炎病例12例,男8例,女4例,年龄范围6~51岁,平均年龄21岁。所有患者在发病后1周内均行胸部X线检查及胸部CT扫描。结果:本组病例中,出现临床症状1~4天胸片像表现为阳性者10例,占83%;4~8天胸片像上为阳性者2例,占17%。多数患者的病灶多发。进展期病变发展迅速,常累及多部位及双肺。好转期一般在7~14天。结论:甲型H1N1流感病毒性肺炎主要影像学表现为:①病变位于肺的外周多,多部位受累,双肺各叶均可发生,以双肺下叶为著;②病变多发,大小不等;③早期最常见的影像学表现为斑片状磨玻璃影;④病变变化迅速。     

聚合酶螺旋反应快速检测甲型H1N1流感病毒

目的 建立利用聚合酶螺旋反应(PSR)快速检测甲型H1N1流感病毒的方法.方法 针对甲型H1N1流感病毒的特异性HA基因设计了6套引物,通过实时浊度法和显色法两种方法判断结果.结果与结论 从6套引物中筛选出了最佳引物,并确定最佳温度为65℃;进一步实验表明最佳引物能特异性地检测H1N1病毒,与14种其他呼吸道病原核酸无交叉反应,敏感性达到100拷贝,与PCR敏感性一致.所建立的方法简单快速、特异性强、敏感性高,适合现场和基层单位应用推广.     

甲型H1N1流感81例临床特征

目的：描述甲型H1N1流感的临床特征。方法：回顾性分析81例甲型H1N1流感患者的病史、临床表现、实验室辅助检查及临床转归。结果：75.3%的患者为青少年学生,发热（100%）、咳嗽（74.1%）和咽痛（37.0%）是其主要症状。体征可见咽部充血（96.2%）和扁桃体肿大（40.7%）。67.9%的患者表现为外周血中性粒细胞百分比增加,而出现肺部X线异常的比例低（7.4%）。96.3%的患者未经抗病毒治疗临床治愈。结论：此次甲型H1N1流感的临床表现与普通季节性甲型流感类似。对甲型H1N1流感的抗病毒治疗共识值得进一步商榷。     

人禽流感HSN1病毒HA1蛋白抗原表位及主要免疫功能区特征的研究

目的 确定人禽流感H5N1病毒HA1蛋白的单克隆抗体(McAb)抗识别表位及主要免疫功能区.方法 将16条人禽流感H5N1病毒HA1基因重叠片段克隆入真核表达载体pDisplay,构建表达HA1蛋白的重叠肽段重组质粒,转染HeLa细胞后制备细胞抗原片.利用灭活的H5N1病毒免疫BALB/C小鼠,制备病毒特异单克隆抗体(单抗)和多克隆抗体(多抗),采用间接免疫荧光法分析单抗、多抗与HA1肽段的反应性.确定HA1蛋白的单抗识别表位及主要免疫功能区.结果 成功克隆、表达了16条不同长度的H5N1肽段,并制备、获得30株分泌抗H5N1型禽流感病毒McAb的杂交瘤细胞株,其中18株为抗HA的单抗,18株中有6株单抗具有中和病毒活性.利用表达的H5N1肽段对单抗识别表位进行分析,初步确定3B5、3D1、3132、M6、M3等18株McAb识别表位区域分别位于氨基酸残基aa133-143、aa155-165、aa166-196、aa166-176、aa34-66及aa296-328之间,其中3B5、3D1、3B2、M6等McAb识别表位为序列依赖型表位,M3和M7等具有中和活性的McAb识别表位为构象性中和表位.结论 利用制备的H5N1病毒特异性McAb和表达的病毒HA1蛋白重叠肽抗原,初步确定了18株抗HA McAb的识别表位和HA1蛋白的主要免疫功能区,为进一步研究开发新型疫苗提供了重要理论依据.     

新甲型H1N1流感病毒及疫情分析

新甲型H1N1流感疫情正在从北美洲向全球逐步蔓延,引起了全世界的关注。本文介绍了此次新甲型H1N1流感的流行概况及特点,分析了病毒的遗传学特征,并尝试性地对新甲型H1N1流感的国际流行态势及对我国的影响进行了初步评估。初步分析认为,新甲型H1N1流感病毒极有可能引发一次全球范围的流感大流行,但其对人类健康造成的危害程度会比1918、1957和1968年3次流感要低;在中国不可避免地会出现一些散发病例甚至小规模的局部暴发流行,但近期造成大规模暴发流行的可能性较小。     

甲型H1N1流感病毒肺炎的CT表现

目的：探讨甲型H1N1流感病毒性肺炎CT表现,提高对该病的认识。方法：回顾性分析34例经临床、实验室证实的甲型H1N1肺炎胸部CT表现。结果：34例中累及两肺5个肺叶17例,占50%;累及4个肺叶3例,占8.8%;累及3个肺叶6例,占17.6%;累及2个肺叶5例,占14.7%;累及1个肺叶3例,占8.8%。单纯毛玻璃样阴影15例,占44.1%,表现为斑片状及云雾样淡薄影,边缘模糊,病灶中心密度相对较高,可见血管纹理通过;单纯实变阴影5例,占14.7%,表现为团片状、斑片状及棉球样影,密度较高;毛玻璃影与实变阴影同时存在14例,占41.2%,表现为云雾样淡薄影、棉球样实变影同时存在,肺周边区域及下叶基底部的病灶密度高于其它部位。实变阴影中可见支气管充气征12例,占35.3%;病灶内合并网格样改变19例,占55.9%;小结节影3例,占8.8%;支气管壁增厚3例,占8.8%;两侧胸腔少量积液1例,占2.9%。结论：甲型H1N1肺炎病变分布范围广,变化快,CT表现具有一定特征性。     

H5N1型流感病毒M1蛋白的基因克隆与表达

目的：克隆、表达A型H5N1流感病毒基质蛋白M1,为研制新的流感快速检测方法奠定基础。方法：应用PCR方法扩增M1的全基因序列,克隆至PET32a载体上,经测序分析确认后,转化感受态大肠杆菌BL21（DE23）,经IPTG诱导表达M1蛋白后,对其表达产物进行SDS-Page和Western Bloting分析。结果：SDS-Page电泳显示M1蛋白在BL21（DE23）大肠杆菌中成功表达,Western Bloting分析显示表达产物可以与M1多克隆抗体发生特异性反应。结论：成功克隆和表达了流感病毒H5N1的基质蛋白M1,为进一步制备高滴度单克隆抗体、研制新的流感检测方法奠定了基础。     

H1N1 pneumonia: our experience in 50 patients with a severe clinical course of novel swine-origin influenza A (H1N1) virus (S-OIV)

Purpose

The authors reviewed chest radiographs (CXR) and thin-section computed tomography (CT) findings of pulmonary complications in a selected population of 50 consecutive patients with severe novel swine-origin influenza A (H1N1) virus (S-OIV) pneumonia who were seen at the Subintensive Respiratory Unit (UTSIR) and at the Intensive Care Unit (ICU) at Monaldi Hospital, Naples, Italy.

Materials and methods

CXR and CT findings of 50 patients who fulfilled the World Health Organisation (WHO) criteria for S-OIV infection were reviewed by four radiologists. The final study group of 50 patients was divided into two subgroups on the basis of clinical course: group 1 consisted of 42 patients requiring noninvasive mechanical ventilation and admitted to the UTSIR; group 2 consisted of eight patients who required ICU admission and extracorporeal membrane oxygenation or advanced mechanical ventilation from October 2009 to December 2009. All patients underwent CXR and thinsection multidetector CT (MDCT) scan; the initial and follow-up radiographs and CT scans were evaluated for the presentation and follow-up pattern (consolidation, groundglass opacities, nodules, reticulation), distribution and extent of abnormality.

Results

All patients had radiological signs of pulmonary involvement. Ground-glass opacity and consolidation, which was mainly peripheral, was the most frequent finding. In three patients, we report for the first time in viral pneumonia the reversed halo sign. Lesion extent was related to aggressiveness of the illness. More often, both lungs were involved (82%). Thoracic comorbidity was present in 18% of patients; 22% of patients was obese, and in this group, the clinical course was more aggressive than in the others with the same lesion extent at imaging. Furthermore, superinfection led to worsening of the clinical conditions.

Conclusions

The most common CXR and CT findings in patients with S-OIV infection were unilateral or bilateral ground-glass opacities with or without associated focal or multifocal areas of consolidation. On MDCT, ground-glass opacities and areas of consolidation had a predominant peribronchovascular and subpleural distribution, resembling organising pneumonia; they progressed to bilateral extensive airspace disease in severely ill patients.     

甲型H1N1流感胸部影像学表现

甲型H1N1流感是2009年在全球爆发流行的呼吸道传染病.引起胸部异常者少见但病人症状较重.主要胸部影像学表现为肺内磨玻璃影和实变影.胸部X线摄影和CT是常用检查手段.就2009年H1N1流感的胸部影像学表现进行综述.     

甲型H1N1流感流行期间不明原因肺炎的CT影像表现

目的:分析甲型H1N1流感流行期间不明原因性肺炎CT影像学特点,指导临床对甲型H1N1流感肺炎的初步诊断.方法:对甲型H1N1流感流行期间因不明原因肺炎住院的患者CT影像表现进行了回顾性分析,CT片分析由两位观察者分别独立进行.临床资料利用SPSS 13.0进行统计分析.结果:甲型H1N1流感肺炎最常见的CT表现为双侧周边磨玻璃密度影,多沿胸膜下分布及围绕肺门区分布.可见①磨玻璃密度影、肺泡渗出、斑片影及肺实变影;②病灶侵犯肺组织广泛,表现为多叶多段的两肺广泛受累;③肺实质、肺间质及胸膜受累可同时存在.结论:影像学检查与诊断仍然是甲型H1N1流感流行期间不明原因性肺炎的临床诊断、鉴别诊断、疗效评价及预后分析的重要手段.其胸部CT影像学特点对甲型H1N1流感肺炎的诊断具有提示作用.     

High-resolution computed tomography findings of influenza virus pneumonia: a comparative study between seasonal and novel (H1N1) influenza virus pneumonia

Purpose  

To evaluate the high-resolution computed tomography (HRCT) findings of novel influenza virus (n-IFV) pneumonia and compare them with the findings for seasonal (s-IFV) pneumonia.     

儿童甲型H1N1流感胸部X线表现

目的 分析儿童甲型H1N1流感患者胸部X线表现特点.方法 回顾性分析67例确诊甲型H1N1流感患儿胸部X线表现,所有患儿于症状出现后3～8 d行仰卧位胸部X线摄片,并对患儿行X线随访.结果 53例甲型H1N1流感患儿病灶分布于肺部双侧,呈多发病灶;7例分布于单侧;7例X线表现正常;42例X线表现为两肺下野斑片状影,10例病灶有融合;7例患儿表现为大叶性肺炎,随访发现1例出现局灶性肺间质增生;11例患儿表现为两肺弥漫性大片状实变影,随访均可见肺间质增生.结论 儿童甲型H1N1流感胸部X线多表现为两肺下野斑片状阴影,其次为两肺弥漫大片状实变影;后者随访可见原病变处有肺间质增生.

Abstract：

Objective To assess the chest X-ray radiographic findings in children with influenza A (H1N1) virus infection. Methods The chest X-ray radiographies in 67 children with influenza A(H1N1) virus infection were reviewed in this study. The chest radiographies were obtained 3-8 days after the onset of symptoms and for the follow-up. Results The abnormalities were bilateral in 53 patients and unilateral in 7 patients. The predominant radiographic findings were bilateral patchy consolidation (n=42) with rapid confluence in 10 patients, lobular consolidation (n=7) with interstitial hyperplasia in 1 patient 3 month later, diffuse consolidation (n=11) with interstitial hyperplasia in all patients after 3 month. ConclusionThe predominant chest X-ray radiographic findings are bilateral patchy consolidation and diffuse consolidation with interstitial hyperplasia afterward.     

科学应对甲型H1N1流感

甲型H1N1病毒在世界范围内的广泛流行引起了WHO的高度重视,疫情警告级别一度从3级提高到4级,又提高到5级.本文从流感病毒的发现、历史上流感大流行的有关情况出发,介绍此次甲型H1N1流感疫情的科学应对方法 ,包括甲型H1N1病毒的传播及甲型H1N1流感的临床表现、治疗、主要防控措施等.     

Thoracic CT findings of novel influenza A (H1N1) infection in immunocompromised patients

The goal of this study is to describe the spectrum of initial and follow-up CT findings of novel influenza A (H1N1) infection in a series of immunocompromised patients. Eight immunocompromised patients with documented novel influenza A (H1N1) had CT imaging at our institution between May 2009 and August 2009. A total of 20 CTs (initial and follow-up) were reviewed for the presence, severity, and distribution of the following: ground glass opacity, consolidation, interlobular septal thickening, mosaic perfusion, airway wall thickening, airway dilatation, nodules, cysts, pleural effusion, pericardial effusion, lymphadenopathy, and air trapping. The most common findings were airway thickening/dilatation, peribronchial ground glass opacity, centrilobular nodules, and tree-in-bud opacities. Peripheral consolidation involving the lower lobes was also a common pattern. Findings frequently involved all lobes and were closely associated with either large or small airways. Two patients presented with atypical CT findings including focal lobar consolidation and patchy lower lobe consolidation with soft tissue centrilobular nodules. Most survivors showed near complete resolution of findings within 35 days. CT scans in immunocompromised patients with novel influenza H1N1 commonly show a strong airway predominance of findings or peripheral areas of consolidation involving the lower lobes. A subset of patients with novel influenza A (H1N1) will show findings not typical of viral infection.