献血/浆人群中人细小病毒B19的亲缘地理学与群体遗传学研究

Human parvovirus B19 (B19V) is a small, nonenveloped, single strand DNA virus with a genome size of 5.6kb belonging to the genus Erythrovirus of the family Parvoviridae. B19V is the most commonly responsible for mild disease, which includes erythema infectiosum (fifth disease), aplastic crisis, chronic pure red cell aplasia, foetal hydrops and foetal death. The virus is also associated with anemia in immunocompromised patients, arthropathies, hepatitis and a variety of other syndromes and diseases. B19V can be transmitted through blood or blood products, for example, via transfusion or B19V-contaminating pooled plasma derived factor FIII. As a result, although the screening for B19V in blood and blood products is not mandated, some western contries have already implanted B19V screening as a routinely test for blood donation. European Pharmacopoeia and Food and Drug Administration launched suggestions to limit the potential B19V burden in plasma pools during the production and a limit of <10-4 IU/mL was imposed for B19V in inactivated plasma. However, there is no routinely test of B19V for Chinese blood/plasma donors. Recently, B19V was classifyied into 3 genotypes. And all the subtypes in the most prevalent B19V genotype I including B19-1A, B191B, and Xi'an Group which was named basing on our research were distributed in China with an abundant genetic variation. In this research, the sero-prevalence of B19V antibodies as well as the prevalence/incedence and viral load of B19V DNA in Chinese blood/plasma donors in 9 donation sites will be determined and the combined data will be evaluated by characteristics of demographics and geographics. After amplification and sequencing, a large number of resulted B19V sequences will be aligned with the homologous sequences downloaded from GenBank which are distributed in various places around the world representing different genotypes and subtypes. Afterwards, the phylogenetic position of the B19V positive samples from the different locations will be allocated in the evolutionary tree and the B19V genotypes and subtypes distributed in China with the prevalence status in different places will be revealed. Then, phylogeography study will be implemented to discorver the distribution and migration pattern and to reveal the reason of rich genetic diversity of B19V in China. The genetic structure and historical demographics in closely phylogenetic related B19V in different localities will be investigated by population dynamics and molecular clock study under certain evolutionary models and rates. We anticipate that, according to this investigation, the results of this study may provide a basis for B19V prevention , control and haemovigilance for the different donation sites, and offer new insights to elevate blood safety in China.

人细小病毒B19（B19V）能通过输血和血液制品传播对人造成危害，但我国没有开展相应的血液检测。B19V基因型I的所有型别：B19-1A、B19-1B和由本课题组发现的B19V西安组病毒，在我国都有分布且遗传多样性丰富。本研究将大量采集来自9个地区的献血者和献浆者的血液样本，检测其中B19V的流行率和新发率，结合人口统计学信息分析B19V流行情况的地理和人口特征差异；通过测序获得B19V阳性样本的DNA序列，分析各地B19V的系统发育位置；结合亲缘关系和地理分化，分析B19V在不同地域的分布迁移模式及其具有丰富遗传多样性的原因；对亲缘关系较近的B19V进行群体遗传分析，探讨不同病毒群体的遗传结构和历史群体动力状态；应用分子钟，推断B19V在各地的起源分化和扩张爆发时间，以及相应的分子进化模式。本课题所得结论能为各地采供血系统针对不同群体和地区特征的B19V，提出相应的防控和预警策略。

人细小病毒B19（Human parvovirus B19, B19V）属于细小病毒科，嗜红细胞病毒属。B19V可通过血液或血液制品传播。为了解B19V对中国输血安全的威胁情况，课题组采集来自绵阳市中心血站，重庆血液中心，洛阳市血液中心，乌鲁木齐血液中心等地的正常无偿献血员单人份血浆各3000余份；成都蓉生药业有限责任公司、四川远大蜀阳药业股份有限公司和贵州泰邦生物制品有限公司的下属浆站献浆员单人份血浆各2000余份，进行系统的流行病学研究。课题组将献血员的血清/血浆样本，按照年龄、性别、民族、血型、婚姻状况、教育程度、职业等特征等人口统计学信息进行归类。通过冷链运输，血液样本被运送至中国医学科学院输血研究所输血传染病研究平台。本课题组开展提取样本 DNA，部分样本 B19V的抗体、 qPCR和巢式 PCR检测。课题组所开发的qPCR检测体系，检测下限为5拷贝/反应体系。B19V在我国5个采供血机构的流行率分别为重庆0.37%、洛阳0.16%、乌鲁木齐2.42%、绵阳0和柳州0.60%，病毒载量（2.66×102-7.2×104）cp/mL，病毒载量中位数为5.65×102 cp/mL；重庆与洛阳、乌鲁木齐、柳州地区比较，B19V的DNA流行率具有显著性差异（P＜0.01）；5地区抗-B19V IgG的检出率具有显著性差异（P＜0.01），5地区抗-B19V IgM的检出率不具有显著性差异（P>0.05）。同时，本研究发现共有16名献血者存在B19V、CMV、HSV交叉感染的情况，占0.42%，实验结果结合献血者样本的个人信息进行人口统计学信息分析。交叉感染的阳性献血者在性别、年龄、职业、婚姻和不同受教育程度中均不具有显著性差异（P>0.05）。然而由于B19V感染者的病毒载量较低，本研究没有成功扩增出感染献血者的B19V全基因组。通过本研究显示，B19V对中国血液安全的威胁程度有限，感染者病毒载量均较低，中国暂不存在由B19V造成的血液安全威胁。

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