抗诺如病毒药物筛选的相关基础及其有效方药的文献研究
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摘要
诺如病毒(NoV)是全球范围内引起成人和儿童非菌性急性胃肠炎暴发流行的重要病原体,同时也是婴幼儿散发性胃肠炎的常见病原(仅次于轮状病毒),无论发达国家还是发展中国家,NoV都有很高的感染率。美国疾病控制中心(CDC)曾经估计,美国每年有至少2300万例NoV导致的胃肠炎病例(约占其人口总数的10%),占到所有已知病原体导致疾病总数的60%,有超过73%到95%的传染性胃肠炎暴发是由NoV造成的,每年因NoV胃肠炎的治疗费用和间接经济损失达3.5至7.5亿美元。据前些年统计,在我国,腹泻病是位居第二位的对小儿威胁较大的常见多发病,其中病毒性腹泻占40%以上,由NoV引起的腹泻估计占1／3。实际上,过去由于未列入常规检测或报告项目,对该病毒感染和发病的的危害仍被严重低估。
NoV基因组属于单股正链RNA,全长约7.5-7.7kb,包括3个开放阅读框(ORFs)。ORF2编码的含540个氨基酸的主要衣壳蛋白VP1为NoV抗原主要决定簇,同时负责识别结合受体,近年来已成为NoV的主要研究对象。NoV具有高度遗传多样性,不同国家、社区同时可能存在不同的NoV毒株流行。自20世纪90年代中期首次确认GⅡ-4型NoV毒株导致绝大部分NoV胃肠炎暴发和世界性大流行后,NoV GⅡ-4型毒株一直为为全球优势流行株。我们课题组南方医科大学公卫学院流行病学系自2002年以来对广东省多地NoV感染进行了前瞻性流行病学研究,结果表明NoV GⅡ-4型为主要流行株,占到总数的82.9%,该结果得到了北京、长春、济南、兰州、福州等地NoV研究的证实。
由于缺乏细胞培养系统,亦无合适的动物感染模型,一直以来抗NoV胃肠炎的药物研究进展缓慢,迄今为止,尚缺乏针对NoV胃肠炎的有效抗病毒药物和疫苗。近年来随着国外学者对NoV基因组结构和发病机制研究的日益深入,发现了一些具有前景的抗NoV药物靶标,如：通过多糖受体阻断病毒与宿主细胞相互作用、抑制病毒蛋白水解酶和多聚酶作用和抑制病毒复制等方面。这些进展为抗NoV性胃肠炎的药物研究提供了新思路。
本研究拟在前期工作基础上,以我国NoV流行优势株GⅡ-4型毒株为主要研究对象,以NoV入侵人体的主要致病蛋白VPl及其所识别的人类组织血型抗原(HBGAs)受体为研究切入点,克隆GⅡ-4型NoV ORF2基因,利用杆状病毒表达系统(Bac-to-Bac)表达纯化GⅡ-4型病毒样颗粒(VLP),在此基础上,采用凝集抑制实验法测定唾液HBGAs受体分型(A、B、H),建立一种体外NoVVLPs结合唾液HBGAs受体的酶联免疫(EIA)药物筛选模型。
中医药治疗病毒性腹泻历史悠久、经验丰富、疗效肯定,且自成体系。近年来中医药界为寻求有效的疗法进行了多方探索,各地涌现出大量自拟新方,经临床和实验证实具有独特的优势和广阔的开发前景。本研究通过收集中医药治疗病毒性腹泻的现代有关文献,试图通过对病毒性腹泻有效方剂之基本结构和核心药物的深入分析,筛选出具有研究前景的候选药物,并拟定更加合理和可行的中医治疗方案,以期为利用此模型进行针对性的高通量抗NoV中药化合物筛选奠定文献基础。
一、GⅡ-4型诺如病毒ORF2基因的克隆与序列分析
从三株GⅡ-4型NoV粪便中(GZ-55, GZ-121, JM-482)提取病毒RNA,经逆转录后,以cDNA为模板,PCR扩增出完整ORF2基因,分别与PMD18-T载体连接,得到重组质粒PMD18-T-GZ55 ORF2, PMD18-T-GZ121 ORF2, PMD18-T-JM482 ORF2。DNA测序后进行全部核苷酸、氨基酸序列分析比较和进化树分析。结果表明：根据GenBank公布的GⅡ-4型NoV保守序列设计合成特异性引物,3株GZ-55, GZ-121, JM-482 NoV粪便标本通过RT-PCR均得到约1.7kb分子量大小ORF2区域片断,其长度与预计相符。重组质粒行BamHⅠ和HindⅢ双酶切后PCR扩增出完整ORF2阅读框序列(约1.62kb)。利用DNAstar软件分析该3株NoV ORF2序列,其中GZ55、GZ121 ORF2共1620个碱基编码539个氨基酸,JM482 ORF2共1623个碱基编码540个氨基酸。GZ55、GZ121和JM482与VA387氨基酸同源性分别为92%、93%、93%。经与Genbank代表性NoV GⅠ、GⅡ的ORF2氨基酸比较分析,结果表明：NoV ORF2包括2个主要区域,N端结尾的壳区域(S区域)和C端结尾的突出区域(P区域)；其编码的氨基酸序列存在3个相对保守序列：S区与P区结合部位的FLVPPTVE序列、P1亚区KT或RT氨基酸序列、P区域C末端保守精氨酸R簇。结合文献分析这些保守序列均具有不同的重要作用：含FLVPPTVE铰链的P二聚体和HBGAs受体结合力低而不含铰链的P颗粒结合HBGAs受体能力非常强：P1亚区KT或RT氨基酸序列为胰岛素酶切位点；C末端保守精氨酸R簇在识别受体过程是必需的。
二、ORF2基因在昆虫细胞中表达形成病毒样颗粒的研究
用限制性内切酶BamHⅠ和HindⅢ将质粒PMD18 -T- GZ121 ORF2酶切,回收酶切的目的基因ORF2片段,再与用BamHⅠ和HindⅢ双酶切的线性化载体PFastBacTM1连接,获得重组转座载体pFastBac-GZ121 ORF2,将其转化大肠杆菌DH10Bac,得到重组杆状病毒穿梭质粒rBacmid-GZ121 ORF2,经用琼脂糖凝胶电泳和PCR鉴定后,将其转染昆虫Sf9细胞,从培养上清中获得重组杆状病毒rBac-GZ121 ORF2,以之重新感染新鲜的Sf9细胞表达外源蛋白。对细胞上清进行SDS-PAGE和Western blot分析,发现重组病毒rBac-GZ121 ORF2表达了分子量约为58KDa的NoV衣壳蛋白,大小与VLP相符,说明杆状病毒携带的NoV衣壳蛋白基因ORF2在昆虫细胞中得到成功表达。对细胞上清采用不连续蔗糖密度梯度超速离心,纯化出VLP并经负染电镜得到证实,其直径约为30nm,形态和结构与野生型NoV相同。为进一步研究VLP的各种特性打下了物质基础。
三、病毒样颗粒结合唾液HBGAs受体的检测及意义
收集健康志愿者唾液标本,采用凝集抑制实验法检测唾液中HBGAs血型物质,用EIA法检测NoV-VLP与HBGAs的结合特性。结果显示,63份唾液标本检出O型21例(占33.3%),B型14例(占22.2%),A型和非分泌型各13例(分别占20.6%),AB型2例(占3.2%)。NoV-VLP与A、B、O型均能结合(OD值>2.5),与非分泌型唾液不结合(OD值-0.3),结合方式与rVA387相一致,其与受体结合的特异性也再次表明我们成功在昆虫细胞中表达了VLP。针对NoV结构蛋白VPl的HBGAs受体阻断剂的筛选和开发具有更为广阔的研究前景和现实意义。本研究建立的NoV-VLP和HBGAs受体结合模型,重复性好,且具有快速经济等优点,为开发我国人群NoV暴发的防治药物提供了理想筛选模型。
四、中医药抗病毒性腹泻有效方药的筛选和评价
以中国生物医学文献光盘数据库(CBMdisc)和中国学术期刊全文数据库(CNKI)1999年1月—2008年12月所收集的文献为资料范围,以(病毒性腹泻or秋季腹泻or急性胃肠炎)and(中医or中药or中成药or中西医结合or经验报道)为计算机检索式,从文献数据库中共检索到文献374篇,进一步评定与筛选剔除不合格文献,最终收集到符合纳入和排除标准的合格文献158篇,每篇文献中关于中药组方的描述均作为一条记录。
通过对组方的用药频次处理,发现中医药抗病毒性腹泻的处方用药主要有如下特点：(1)重视经典方剂的运用：明确提到以经典方剂(传统成方)加味治疗的文献47篇,占总数的34.1%。核心方剂是五苓散、葛根芩连汤、泻心汤、藿香正气散、理中汤和七味白术散。(2)用药集中：主要集中在云苓、白术、葛根、藿香、苍术、黄芩、车前子等31味核心药物,占药物出现总频次的78.5%。药物类别主要集中在补虚药、利水渗湿药和化湿药等十大类。(3)健脾渗湿并重：补虚药用药频次最多,且主要集中在健脾益气药(白术、党参、山药、扁豆)；利水渗湿药仅居其次,集中在云苓、车前子、泽泻、猪苓、薏苡仁。“以方测证”,可见脾虚湿盛是病毒性腹泻的主要病机。(4)兼顾病因,加减化裁,忌闭门留寇：除注重健脾渗湿外,化湿药、清热药、消食药、解表药、温里药比重相对平均,证明致泄病因主要集中在湿、热、食滞、寒几类。临床根据兼夹不同,可随证加减,或清化湿热、或温化寒湿、或疏表、或消导。收涩药出现频次仅居第八位,表明多数医家不主张骤用补涩,以防闭门留寇。
通过对治疗病毒性腹泻有效方药的筛选和分析,特拟定优化组方用药方案如下：炒白术10g、云苓10g、藿香10g、煨葛根10g、车前子10g、乌梅5g。
Noroviruses (NoV) are a leading cause of epidemics of acute nonbacterial gastroenteritis, affecting people of all ages worldwide. NoV infection with high infection rates exist in populations both in developing and developed countries. The viruses are highly contagious, usually resulting in large outbreaks in crowded communities or institutions such as schools, restaurants, hospitals, child care centers, nursing homes for the elderly, cruise ships, and military settings. It was estimated that 10% of the population of United States experienced NoV gastroenteritis each year (23 million cases); and NoV accounts for a substantial extent of disease and potential economic loss (35-75 million dollars), particularly to the health service where a large proportion of outbreaks occur. In China, acute gastroenteritis (AGE) is one of the most common illnesses of children, as well as in other developing countries. In family settings gastroenteritis is second in frequency only to the common cold. However, there are only few reports on the prevalence of NOV in China. So far, AGE caused by NoV infections were not a routine surveillance project, just classified into C grade infectious diseases as other infectious diarrhea, in China. And the role of NoV infections was over underestimated.
NOV are small (～38 nm in diameter), nonenveloped, singlestranded, positive-sense RNA viruses belonging to the family Caliciviridae. The NOV genome encodes three open reading frames (ORF), one of which, ORF-2, encodes one major structural protein of～60 kDa that spontaneously forms virus-like particles (VLPs) when expressed in baculovirus or in other expression systems. These VLPs are morphologically and antigenically indistinguishable from the native forms of viruses found in human stools, providing valuable materials for the development of immunological assays, for the study of virus-receptor host interaction.
NoV are difficult to study, because there is no cell culture or animal model available for them, and the disease is difficult to control because of a lack of vaccines or effective antivirals against NoV. It is therefore a public health priority to develop an effective strategy for the prevention and treatment of NoV infection. Despite the difficulty in cultivating noroviruses, significant advances in understanding the genomic structure, individual viral proteins, RNA replication strategy, and virus-host interaction of the virus have been made. These advancements provide new strategies in the development of antiviral agents against norovirus, including the inhibition of viral attachment to host cells through carbohydrate receptors, inhibition of viral protease and polymerase functions, and interference in viral replication.
GII-4 norovirus strain now was recognized as the predominant strain circulating around the world in last 20 years. In this study, VLPs from a GⅡ-4 NoV, which was identified as the prevalence strain in China in our former studies, were expressed in insect cells. EIA methods for VLPs binding to HBGAs receptor were also established. These works will provide basis for future drug screening.
Chinese medicine had a long history for viral diarrhea treatment. More and more prescriptions were explored in these years with good effect. In the present study, herb and prescriptions were screened by document study as future Chinese medicine candidate.
1. Clone and Sequence Analysis of the ORF2 Gene of GⅡ-4 norovirus
RNA was extracted from fecal sample. After transcription and PCR, the ORF2 gene was cloned in sample T-vector. Sequences were analyzed by using DNAstar software and compared with prototype sequences retrieved from the NCBI database by Clustal X and Treeview software. Exact whole ORF2 gene of norovirus GⅡ-4 strain was obtained which code 54 KD amino acid. The capsid region consists of two major domains, the shell (S) and the protrusion (P) domains, which are linked by a short hinge. The S domain forms the interior shell of the capsid, while the P domain builds up arch-like structures extending from the shell. Three ORF2 gene of norovirus GⅡ-4 strain was obtained and strains in this study showed 92-93% homology with VA387 strains. And there existed 3 conserved regions in ORF2 region:a short hinge (FLVPPTVE) between P and S domain; the trypsin cleavage sites (KT or RT amino acid) and a highly conserved arginine (R) cluster at the C terminus of the P domain, which is critical for receptor binding.
2. Expression of NoV capsid protein (VLPs) in insect cells
GZ121 NoV ORF2 gene amplified by PCR by using PMD18-T plasmid, was inserted into baculovirus expression system, constructed a recombinant vector, pFastBac-GZ121 ORF2. After the transposition in DH10Bac, recombinant baculovirus rBacmid-GZ121 ORF2 was gained. After the transfection to Sf9 cells, rBac-GZ121 ORF2 was obtained in supernatant culture. VLPs expression were confirmed by using SDS-PAGE and Western blot and showed an expected molecular mass about 58KDa. And the expressed VLPs had a 30nm diameter under Electronic Microscope. It indicated the successful expression VLPs in insect cells, which will provide material basis for further studying the characteristic of NoV VLPs.
3. GZ121 VLPs bind to Saliva HBGAs receptor and its significance
Saliva specimens were collected from 63 volunteers; HBGAs antigens types were identified by using agglutination inhibition assays; the binding patterns of NoV to HBGAs antigens were identified by EIA. Among 63 volunteers, O-antigen accounted for 33.3%, B for 22.2%, A and non-secretor for 20.6% and AB for 3.2%, respectively. GZ121 VLPs can bind to all secretors (OD>2.5), but not to all nonsecretors (OD≦0.3). These results also confirmed our successful expression VLPs in insect cells. We established experimental methods on NoV binding to saliva HBGAs antigen; it will help us to explore the host adaptation character, virus evolution and epidemiology of NoV; it will also help us to establish technique platform of anti-NoV drugs screening, and lay the basis for NoV prevention and therapy in Chinese population.
4. Screening and evaluation of effective herbs and formulae for viral diarrhea
We used words (viral diarrhea, autumn diarrhea, acute gastroenteritis, and Chinese medicine, Chinese herb, Chinese medicine product, integrated traditional chinese and western medicine and experience report) as key words and did search documents in CBMdisc and CNKI from Jan 1999 to Dec 2008; and we obtained 374 documents. After eliminating unqualified documents,158 documents were selected to our study. The herbs or formulae from one document saved as a record.
After the frequency study of the herbs and formulae in these documents, some characteristics of herbs and formulae were found:(1) emphasizing on the use of classic traditional formulae. (2) Emphasizing on some herbs; (3) equally focusing on invigorating the spleen for eliminating dampness; (4) also focusing on the cause of the disease.
Combining with the screening and analysis of the effective herbs for viral diarrhea, we make the essence prescription:baked white atractylodes rhizome (10g), Poria from Yunnan (10g), Agastache rugosus(10g), roasted Radx Puerariae(10g), plantago seed(10g)，dark plum(5g).
NoV基因组属于单股正链RNA,全长约7.5-7.7kb,包括3个开放阅读框(ORFs)。ORF2编码的含540个氨基酸的主要衣壳蛋白VP1为NoV抗原主要决定簇,同时负责识别结合受体,近年来已成为NoV的主要研究对象。NoV具有高度遗传多样性,不同国家、社区同时可能存在不同的NoV毒株流行。自20世纪90年代中期首次确认GⅡ-4型NoV毒株导致绝大部分NoV胃肠炎暴发和世界性大流行后,NoV GⅡ-4型毒株一直为为全球优势流行株。我们课题组南方医科大学公卫学院流行病学系自2002年以来对广东省多地NoV感染进行了前瞻性流行病学研究,结果表明NoV GⅡ-4型为主要流行株,占到总数的82.9%,该结果得到了北京、长春、济南、兰州、福州等地NoV研究的证实。
由于缺乏细胞培养系统,亦无合适的动物感染模型,一直以来抗NoV胃肠炎的药物研究进展缓慢,迄今为止,尚缺乏针对NoV胃肠炎的有效抗病毒药物和疫苗。近年来随着国外学者对NoV基因组结构和发病机制研究的日益深入,发现了一些具有前景的抗NoV药物靶标,如：通过多糖受体阻断病毒与宿主细胞相互作用、抑制病毒蛋白水解酶和多聚酶作用和抑制病毒复制等方面。这些进展为抗NoV性胃肠炎的药物研究提供了新思路。
本研究拟在前期工作基础上,以我国NoV流行优势株GⅡ-4型毒株为主要研究对象,以NoV入侵人体的主要致病蛋白VPl及其所识别的人类组织血型抗原(HBGAs)受体为研究切入点,克隆GⅡ-4型NoV ORF2基因,利用杆状病毒表达系统(Bac-to-Bac)表达纯化GⅡ-4型病毒样颗粒(VLP),在此基础上,采用凝集抑制实验法测定唾液HBGAs受体分型(A、B、H),建立一种体外NoVVLPs结合唾液HBGAs受体的酶联免疫(EIA)药物筛选模型。
中医药治疗病毒性腹泻历史悠久、经验丰富、疗效肯定,且自成体系。近年来中医药界为寻求有效的疗法进行了多方探索,各地涌现出大量自拟新方,经临床和实验证实具有独特的优势和广阔的开发前景。本研究通过收集中医药治疗病毒性腹泻的现代有关文献,试图通过对病毒性腹泻有效方剂之基本结构和核心药物的深入分析,筛选出具有研究前景的候选药物,并拟定更加合理和可行的中医治疗方案,以期为利用此模型进行针对性的高通量抗NoV中药化合物筛选奠定文献基础。
一、GⅡ-4型诺如病毒ORF2基因的克隆与序列分析
从三株GⅡ-4型NoV粪便中(GZ-55, GZ-121, JM-482)提取病毒RNA,经逆转录后,以cDNA为模板,PCR扩增出完整ORF2基因,分别与PMD18-T载体连接,得到重组质粒PMD18-T-GZ55 ORF2, PMD18-T-GZ121 ORF2, PMD18-T-JM482 ORF2。DNA测序后进行全部核苷酸、氨基酸序列分析比较和进化树分析。结果表明：根据GenBank公布的GⅡ-4型NoV保守序列设计合成特异性引物,3株GZ-55, GZ-121, JM-482 NoV粪便标本通过RT-PCR均得到约1.7kb分子量大小ORF2区域片断,其长度与预计相符。重组质粒行BamHⅠ和HindⅢ双酶切后PCR扩增出完整ORF2阅读框序列(约1.62kb)。利用DNAstar软件分析该3株NoV ORF2序列,其中GZ55、GZ121 ORF2共1620个碱基编码539个氨基酸,JM482 ORF2共1623个碱基编码540个氨基酸。GZ55、GZ121和JM482与VA387氨基酸同源性分别为92%、93%、93%。经与Genbank代表性NoV GⅠ、GⅡ的ORF2氨基酸比较分析,结果表明：NoV ORF2包括2个主要区域,N端结尾的壳区域(S区域)和C端结尾的突出区域(P区域)；其编码的氨基酸序列存在3个相对保守序列：S区与P区结合部位的FLVPPTVE序列、P1亚区KT或RT氨基酸序列、P区域C末端保守精氨酸R簇。结合文献分析这些保守序列均具有不同的重要作用：含FLVPPTVE铰链的P二聚体和HBGAs受体结合力低而不含铰链的P颗粒结合HBGAs受体能力非常强：P1亚区KT或RT氨基酸序列为胰岛素酶切位点；C末端保守精氨酸R簇在识别受体过程是必需的。
二、ORF2基因在昆虫细胞中表达形成病毒样颗粒的研究
用限制性内切酶BamHⅠ和HindⅢ将质粒PMD18 -T- GZ121 ORF2酶切,回收酶切的目的基因ORF2片段,再与用BamHⅠ和HindⅢ双酶切的线性化载体PFastBacTM1连接,获得重组转座载体pFastBac-GZ121 ORF2,将其转化大肠杆菌DH10Bac,得到重组杆状病毒穿梭质粒rBacmid-GZ121 ORF2,经用琼脂糖凝胶电泳和PCR鉴定后,将其转染昆虫Sf9细胞,从培养上清中获得重组杆状病毒rBac-GZ121 ORF2,以之重新感染新鲜的Sf9细胞表达外源蛋白。对细胞上清进行SDS-PAGE和Western blot分析,发现重组病毒rBac-GZ121 ORF2表达了分子量约为58KDa的NoV衣壳蛋白,大小与VLP相符,说明杆状病毒携带的NoV衣壳蛋白基因ORF2在昆虫细胞中得到成功表达。对细胞上清采用不连续蔗糖密度梯度超速离心,纯化出VLP并经负染电镜得到证实,其直径约为30nm,形态和结构与野生型NoV相同。为进一步研究VLP的各种特性打下了物质基础。
三、病毒样颗粒结合唾液HBGAs受体的检测及意义
收集健康志愿者唾液标本,采用凝集抑制实验法检测唾液中HBGAs血型物质,用EIA法检测NoV-VLP与HBGAs的结合特性。结果显示,63份唾液标本检出O型21例(占33.3%),B型14例(占22.2%),A型和非分泌型各13例(分别占20.6%),AB型2例(占3.2%)。NoV-VLP与A、B、O型均能结合(OD值>2.5),与非分泌型唾液不结合(OD值-0.3),结合方式与rVA387相一致,其与受体结合的特异性也再次表明我们成功在昆虫细胞中表达了VLP。针对NoV结构蛋白VPl的HBGAs受体阻断剂的筛选和开发具有更为广阔的研究前景和现实意义。本研究建立的NoV-VLP和HBGAs受体结合模型,重复性好,且具有快速经济等优点,为开发我国人群NoV暴发的防治药物提供了理想筛选模型。
四、中医药抗病毒性腹泻有效方药的筛选和评价
以中国生物医学文献光盘数据库(CBMdisc)和中国学术期刊全文数据库(CNKI)1999年1月—2008年12月所收集的文献为资料范围,以(病毒性腹泻or秋季腹泻or急性胃肠炎)and(中医or中药or中成药or中西医结合or经验报道)为计算机检索式,从文献数据库中共检索到文献374篇,进一步评定与筛选剔除不合格文献,最终收集到符合纳入和排除标准的合格文献158篇,每篇文献中关于中药组方的描述均作为一条记录。
通过对组方的用药频次处理,发现中医药抗病毒性腹泻的处方用药主要有如下特点：(1)重视经典方剂的运用：明确提到以经典方剂(传统成方)加味治疗的文献47篇,占总数的34.1%。核心方剂是五苓散、葛根芩连汤、泻心汤、藿香正气散、理中汤和七味白术散。(2)用药集中：主要集中在云苓、白术、葛根、藿香、苍术、黄芩、车前子等31味核心药物,占药物出现总频次的78.5%。药物类别主要集中在补虚药、利水渗湿药和化湿药等十大类。(3)健脾渗湿并重：补虚药用药频次最多,且主要集中在健脾益气药(白术、党参、山药、扁豆)；利水渗湿药仅居其次,集中在云苓、车前子、泽泻、猪苓、薏苡仁。“以方测证”,可见脾虚湿盛是病毒性腹泻的主要病机。(4)兼顾病因,加减化裁,忌闭门留寇：除注重健脾渗湿外,化湿药、清热药、消食药、解表药、温里药比重相对平均,证明致泄病因主要集中在湿、热、食滞、寒几类。临床根据兼夹不同,可随证加减,或清化湿热、或温化寒湿、或疏表、或消导。收涩药出现频次仅居第八位,表明多数医家不主张骤用补涩,以防闭门留寇。
通过对治疗病毒性腹泻有效方药的筛选和分析,特拟定优化组方用药方案如下：炒白术10g、云苓10g、藿香10g、煨葛根10g、车前子10g、乌梅5g。
Noroviruses (NoV) are a leading cause of epidemics of acute nonbacterial gastroenteritis, affecting people of all ages worldwide. NoV infection with high infection rates exist in populations both in developing and developed countries. The viruses are highly contagious, usually resulting in large outbreaks in crowded communities or institutions such as schools, restaurants, hospitals, child care centers, nursing homes for the elderly, cruise ships, and military settings. It was estimated that 10% of the population of United States experienced NoV gastroenteritis each year (23 million cases); and NoV accounts for a substantial extent of disease and potential economic loss (35-75 million dollars), particularly to the health service where a large proportion of outbreaks occur. In China, acute gastroenteritis (AGE) is one of the most common illnesses of children, as well as in other developing countries. In family settings gastroenteritis is second in frequency only to the common cold. However, there are only few reports on the prevalence of NOV in China. So far, AGE caused by NoV infections were not a routine surveillance project, just classified into C grade infectious diseases as other infectious diarrhea, in China. And the role of NoV infections was over underestimated.
NOV are small (～38 nm in diameter), nonenveloped, singlestranded, positive-sense RNA viruses belonging to the family Caliciviridae. The NOV genome encodes three open reading frames (ORF), one of which, ORF-2, encodes one major structural protein of～60 kDa that spontaneously forms virus-like particles (VLPs) when expressed in baculovirus or in other expression systems. These VLPs are morphologically and antigenically indistinguishable from the native forms of viruses found in human stools, providing valuable materials for the development of immunological assays, for the study of virus-receptor host interaction.
NoV are difficult to study, because there is no cell culture or animal model available for them, and the disease is difficult to control because of a lack of vaccines or effective antivirals against NoV. It is therefore a public health priority to develop an effective strategy for the prevention and treatment of NoV infection. Despite the difficulty in cultivating noroviruses, significant advances in understanding the genomic structure, individual viral proteins, RNA replication strategy, and virus-host interaction of the virus have been made. These advancements provide new strategies in the development of antiviral agents against norovirus, including the inhibition of viral attachment to host cells through carbohydrate receptors, inhibition of viral protease and polymerase functions, and interference in viral replication.
GII-4 norovirus strain now was recognized as the predominant strain circulating around the world in last 20 years. In this study, VLPs from a GⅡ-4 NoV, which was identified as the prevalence strain in China in our former studies, were expressed in insect cells. EIA methods for VLPs binding to HBGAs receptor were also established. These works will provide basis for future drug screening.
Chinese medicine had a long history for viral diarrhea treatment. More and more prescriptions were explored in these years with good effect. In the present study, herb and prescriptions were screened by document study as future Chinese medicine candidate.
1. Clone and Sequence Analysis of the ORF2 Gene of GⅡ-4 norovirus
RNA was extracted from fecal sample. After transcription and PCR, the ORF2 gene was cloned in sample T-vector. Sequences were analyzed by using DNAstar software and compared with prototype sequences retrieved from the NCBI database by Clustal X and Treeview software. Exact whole ORF2 gene of norovirus GⅡ-4 strain was obtained which code 54 KD amino acid. The capsid region consists of two major domains, the shell (S) and the protrusion (P) domains, which are linked by a short hinge. The S domain forms the interior shell of the capsid, while the P domain builds up arch-like structures extending from the shell. Three ORF2 gene of norovirus GⅡ-4 strain was obtained and strains in this study showed 92-93% homology with VA387 strains. And there existed 3 conserved regions in ORF2 region:a short hinge (FLVPPTVE) between P and S domain; the trypsin cleavage sites (KT or RT amino acid) and a highly conserved arginine (R) cluster at the C terminus of the P domain, which is critical for receptor binding.
2. Expression of NoV capsid protein (VLPs) in insect cells
GZ121 NoV ORF2 gene amplified by PCR by using PMD18-T plasmid, was inserted into baculovirus expression system, constructed a recombinant vector, pFastBac-GZ121 ORF2. After the transposition in DH10Bac, recombinant baculovirus rBacmid-GZ121 ORF2 was gained. After the transfection to Sf9 cells, rBac-GZ121 ORF2 was obtained in supernatant culture. VLPs expression were confirmed by using SDS-PAGE and Western blot and showed an expected molecular mass about 58KDa. And the expressed VLPs had a 30nm diameter under Electronic Microscope. It indicated the successful expression VLPs in insect cells, which will provide material basis for further studying the characteristic of NoV VLPs.
3. GZ121 VLPs bind to Saliva HBGAs receptor and its significance
Saliva specimens were collected from 63 volunteers; HBGAs antigens types were identified by using agglutination inhibition assays; the binding patterns of NoV to HBGAs antigens were identified by EIA. Among 63 volunteers, O-antigen accounted for 33.3%, B for 22.2%, A and non-secretor for 20.6% and AB for 3.2%, respectively. GZ121 VLPs can bind to all secretors (OD>2.5), but not to all nonsecretors (OD≦0.3). These results also confirmed our successful expression VLPs in insect cells. We established experimental methods on NoV binding to saliva HBGAs antigen; it will help us to explore the host adaptation character, virus evolution and epidemiology of NoV; it will also help us to establish technique platform of anti-NoV drugs screening, and lay the basis for NoV prevention and therapy in Chinese population.
4. Screening and evaluation of effective herbs and formulae for viral diarrhea
We used words (viral diarrhea, autumn diarrhea, acute gastroenteritis, and Chinese medicine, Chinese herb, Chinese medicine product, integrated traditional chinese and western medicine and experience report) as key words and did search documents in CBMdisc and CNKI from Jan 1999 to Dec 2008; and we obtained 374 documents. After eliminating unqualified documents,158 documents were selected to our study. The herbs or formulae from one document saved as a record.
After the frequency study of the herbs and formulae in these documents, some characteristics of herbs and formulae were found:(1) emphasizing on the use of classic traditional formulae. (2) Emphasizing on some herbs; (3) equally focusing on invigorating the spleen for eliminating dampness; (4) also focusing on the cause of the disease.
Combining with the screening and analysis of the effective herbs for viral diarrhea, we make the essence prescription:baked white atractylodes rhizome (10g), Poria from Yunnan (10g), Agastache rugosus(10g), roasted Radx Puerariae(10g), plantago seed(10g)，dark plum(5g).
引文
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