E. coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.     

Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase

Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.     

一起诺如病毒性胃肠炎暴发的队列研究

目的调查分析某村一起诺如病毒性胃肠炎暴发的发病原因。方法采用历史性队列研究方法，应用ELISA对标本进行实验室检测。结果该起暴发共报告病例34例，发病与接触腹泻病人（X^2=6．51，P=0．00）、食物暴露于苍蝇（X^2=4．61，P=0．00）及村民暴露于患者呕吐物或粪便1m内（X^2=3．09，P=0．00）有统计学意义上的关联，与饮用水来源（X^2=1．68，P=0．14）没有统计学意义上的关联。在采集的8份肛拭子样本中1份ELISA法检出诺如病毒抗原阳性：4份粪便样本中2份ELISA法检出诺如病毒抗原阳性。结论这是一起由诺如病毒感染所致的急性胃肠炎暴发．密切接触是此次暴发的主要传播途径。     

Seroprevalence studies using a recombinant norwalk virus protein enzyme immunoassay

A recombinant Norwalk virus (NV) protein enzyme immunoassay was used to study the age of acquisition of NV IgG in various populations. In London, England, there was little evidence of infection during the first 2 years of life. However, the prevalence of NV IgG rose steadily throughout the period that children attend school, reaching a peak of 70% in the group aged 11–16 years. High levels of maternal antibody were detected in infants aged <3 months. Comparison of the acquisition of antibodies to three strains of human calicivirus in Japanese children in northern Japan indicated that although the majority had experienced infection with strains Japan and UK1 by the age of 12 years, only 22% possessed antibodies to NV. In Australian aborigines NV infection occurs early in life; by the age of 6 years over 90% of children were seropositive. © 1994 Wiley-Liss, Inc.     

桂林市诺瓦克病毒性腹泻暴发疫情调查

目的 了解诺瓦克病毒性腹泻流行特征,为制定防治对策提供依据.方法 应用现场流行病学方法调查疫情特征、临床表现,采集标本,用逆转录-聚合酶链反应(RT-PCR)进行病原学检测.结果 该疫情共发生病例54例,发病年龄8月86岁;主要临床表现为发热、呕吐、腹痛、腹泻;病程35 d;采集7份患者粪便标本,经RT-PCR检测,4份诺瓦克病毒阳性;进行传染源管理、饮用水消毒、和对村民进行健康教育等综合处理措施后疫情得到有效控制.结论 诺瓦克病毒性腹泻主要临床症状有腹泻、呕吐,少数病例低热,病例一般症状较轻,可自愈,患者粪便样诺瓦克病毒阳性;综合防控措施可有效控制疫情.     

深圳市某校一起诺瓦克病毒性胃肠炎疫情调查分析

目的:调查以发热呕吐、腹泻为主要症状的胃肠炎的病因,查找传染源,以采取相应的控制措施。方法:采用流行病学调查方法对病例进行调查,对病人以及食堂员工肛拭、粪便以及环境样品采用实时荧光PCR进行定性检测。结果:经诺瓦克病毒基因检测,学生样本3例阳性;教师食堂员工样本阴性;环境样本1例阳性。6月15、19日对73名学生食堂员工进行诺瓦克病毒基因检测,分别有7例(9.6%)和5例(6.9%)阳性。25日,再次对73名食堂员工进行检测,全为阴性。通过测序分析比对,从食堂员工以及学生中检测出的诺瓦克病毒为同一型诺瓦克病毒。结论:该校学生胃肠炎是由诺瓦克病毒感染引起,学生食堂员工带毒是引起此次诺瓦克病毒发生流行的传染源。     

反思一起诺瓦克样病毒引起的暴发调查

目的：为更好地处理突发公共卫生事件，提高应急处理与防控能力，总结经验教训。方法：对该起事件作回顾性检视，对不妥当的行为逐一记录并分析其原因。结果：存在问题的有迟报、对病患学生的错误认识、食堂承包者对流行病学调查工作的干扰、样品采集、对食堂的控制、食堂的预防措施、应对媒体等。结论：危险源／传染源未清除、在食堂内采取的预防措施针对性差且存在缺陷与不足，是疫情出现第二个流行高峰、规模扩大、时间延长的直接主要原因。     

Intranasal vaccination with an adjuvanted Norwalk virus-like particle vaccine elicits antigen-specific B memory responses in human adult volunteers

Noroviruses are the most frequent cause of acute gastroenteritis in humans of all ages. No vaccines are currently available. An intranasally delivered Norwalk (NV) virus-like particle (VLP) vaccine was recently shown to be well tolerated, immunogenic and to protect against infection in Phase 1 studies. Here, we examined B memory (B(M)) responses in volunteers who received the highest dosage levels of the NV-VLP vaccine (50 μg and 100 μg). We measured the frequency of NV-specific IgG and IgA-secreting B(M) cells in peripheral blood and the level of antibodies produced by these cells in culture. All subjects immunized with 100 μg of the NV-VLP vaccine and 90% of those who received 50 μg had significant IgA or IgG B(M) responses. The B(M) cell frequencies correlated with serum antibody levels and mucosally-primed antibody-secreting cell responses. This is the first demonstration of dose-dependent, functional B(M) responses in humans immunized intranasally with a NV-VLP vaccine.