2020~2021年广州市售牡蛎中GII型诺如病毒污染情况调查

滤食性牡蛎是食源性诺如病毒传播的重要食品媒介。为了解广州市售牡蛎中的诺如病毒污染水平与遗传多样性特点,合理评估消费风险,本研究于2020年6月至2021年5月期间,每月从当地水产市场随机采集牡蛎样本,采用实验室前期建立的蛋白酶K处理偶联聚乙二醇沉淀小体系法,包括荧光定量RT-PCR和巢式RT-PCR技术检测贝类中病毒的污染量以及基因型分布特点。结果共检测牡蛎110只,GII型诺如病毒阳性检出率为52.7%（58/110）,病毒污染含量范围为1.56×103~1.09×106 copies/g（消化腺）。其中,春夏季节（3~8月）牡蛎中诺如病毒的阳性率为35.7%（20/56）,低于在秋冬季节（9~2月）的阳性率70.4%（38/54）;但不同季节中检出的病毒含量无显著差异,分别为春季（2.69±1.46）×105 copies/g（消化腺）,夏季（1.97±2.16）×105 copies/g（消化腺）,秋季（6.91±6.16）×104 copies/g（消化腺）,冬季为（4.83±2.99）×104 copies/g（消化腺）。部分阳性样本测序分析后显示,除1份为GII.17基因型外,其余均为GII.4基因型（n=13）,与当地的临床流行基因型呈现一致性。本研究显示广州市售牡蛎中仍存在较高的诺如病毒污染水平,需要进一步加强病毒防控工作,尤其提醒消费者在食用牡蛎时需加工充分。     

Detection of noroviruses in shellfish in the Netherlands

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish (n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.     

Enhanced immunomagnetic separation for the detection of norovirus using the polyclonal antibody produced with human norovirus GII.4-like particles

Human norovirus (NoV) is the most common cause of foodborne viral gastroenteritis worldwide. This study was aimed to develop the enhanced immunomagnetic separation (IMS) for effectively concentrating and detecting human noroviruses in food matrix. Virus-like particles (VLPs) were made by integrating NoV GII.4 capsid gene into baculovirus vector. In order to increase the sensitivity and specificity of immunomagnetic complex, polyclonal rabbit antibody against NoV GII.4 capsid was produced and used for producing immunomagnetic beads. IMS, polyethylene glycol precipitation, and ultrafiltration were compared to concentrate NoV spiked in vegetables. IMS was the most efficient method for concentrating NoV. Therefore, IMS developed in this study is the most effective method to concentrate and detect NoV contaminated in produce.     

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.     

Construction and Analytical Application of Internal Amplification Controls (IAC) for Detection of Food Supply Chain-Relevant Viruses by Real-Time PCR-Based Assays

Internal amplification controls (IACs) were constructed for incorporation into real-time nucleic acid amplification assays for bovine polyomavirus, hepatitis A virus, hepatitis E virus, human adenovirus, human norovirus genogroup I, human norovirus genogroup II, murine norovirus and porcine adenovirus. The addition of optimised amounts of IAC into the assays did not affect the limits of detection for each specific target virus. A poorly performed extraction of viral nucleic acids was simulated, and the effectiveness of IACs in identifying failed assays was demonstrated. The IACs constructed in this study can be reliably used in their specific assays to provide a robust control that can be routinely applied in the analysis of foods for viruses.     

Genetic variation in the conservative gene region of Norovirus genogroup II strains in environmental and stool samples

Noroviruses (NoVs) have been one of leading etiological agents for infectious gastroenteritis over the world. Gastroenteritis caused by NoVs is prevalent in winter season, and the contamination of the water environment with NoVs in the epidemic cold season is frequently reported. In contrast, the number of gastroenteritis patients and NoVs in the water environment are reduced during the nonepidemic summer season, and the year-round fate of NoVs has remained to be elucidated. In this study, we collected nucleotide sequences of NoV genogroup II (GII) from domestic sewage, sewage sludge, treated wastewater, river water, and stool samples of gastroenteritis patients in geographically close areas. Phylogenetic analysis of the obtained NoV gene revealed that six out of seven isolates from environmental samples and 10 out of 11 isolates from stool samples belong to genotype 3 (NoV GII.3) or 4 (NoV GII.4), which have been prevalent throughout the world. Genetic distances between the conservative gene region of NoV GII.4 variants implied that genetically diverse strains are likelyto occur in environmental samples. The evaluation of the evolutionary change of NoV gene obtained from environmental samples would make it possible to elucidate the year-round fate of NoVs.     

草莓中诺如病毒和甲肝病毒的多重实时荧光逆转录PCR检测方法的建立

目的建立草莓中诺如病毒GI、诺如病毒GII和甲肝病毒等3种食源性病毒的多重实时荧光RT-PCR检测方法,并应用于实际样品检测。方法对草莓样品进行前处理、病毒富集、病毒RNA提取和纯化后,先采用单重实时荧光RT-PCR进行检测,随后进行多重实时荧光RT-PCR反应条件优化,建立多重实时荧光RT-PCR检测方法并分析其特异性和灵敏度。结果所采用的病毒富集和核酸提取方法可以实现病毒的有效富集和抑制剂的去除,建立的多重实时荧光RT-PCR方法特异性强(100%),对草莓样品中诺如病毒GI、诺如病毒GII和甲肝病毒的检测灵敏度分别为56.2 RT-PCR50/20 g、31.6 RT-PCR50/20 g和31.4 CCID50/20 g。同时对50份样品进行检测,结果均为阴性。结论所建立的检测方法快速、灵敏、特异性强,适用于草莓产品中诺如病毒GI、诺如病毒GII和甲肝病毒的同时检测。