摘要
新发病毒性传染病严重危害人类的健康,影响社会、经济稳定,甚至威胁国家安全,引起全球高度关注。人博卡病毒(Human bocavirus, HBoV)是2005年被发现的一种新病毒,可能与呼吸道感染有关。目前,已经发现了4种HBoV(HBoV1-4),但其致病性均尚未阐明。现有关于HBoV的流行情况研究大多集中于HBoV1和HBoV2,关于HBoV3和HBoV4的报道很少,且4种HBoV在人群中的流行水平尚不清楚。此外,由于HBoV之间具有较高的氨基酸同源性,它们之间的血清学交叉反应尚不清楚,给HBoV的免疫学检测方法研究和评价带来困难。为此,本研究对北京地区急性呼吸道感染儿童中4种HBoV的流行率和健康人群中HBoV1、2、3的血清抗体流行率进行了研究,并对HBoV的主要抗原VP2蛋白的表位进行了筛选。主要结果如下：
一、北京地区人博卡病毒分子流行率调查
在2008年3月至2010年7月间收集在北京儿童医院住院的患急性下呼吸道感染的患儿鼻咽洗液标本1238份,进行HBoV通用PCR检测。结果显示：HBoV总检出率为11.4%,其中HBoV1检出率为10.6%,HBoV2为0.4%,HBoV3为0.4%。无HBoV4检出。HBoV感染主要集中于5岁以下儿童,占HBoV阳性病例数的93.6%。与以往报道类似,与其它呼吸道病毒的共检出率可达85.1%。HBoV检出没有明显的季节差异。此外,2006年3月-2007年11月从北京儿童医院门诊收集患急性胃肠炎的366份儿童腹泻粪便标本,进行HBoV PCR检测。结果显示：HBoV总检出率为12%,阳性病例年龄分布为2个月至24个月(中位数为9个月)。其中HBoV1检出率为2.5%,HBoV2为9.0%,HBoV3为0.5%,无HBoV4检出。HBoV与其它病毒共检出率为79.5%。进化树分析和重组分析结果显示,HBoV3可能来源于HBoV1和HBoV2的基因重组,重组位点位于NS1基因的上游和VP1/2读码框中；HBoV3也可能源于HBoV1和HBo4的基因重组。本研究首次在呼吸道样本中检出HBoV3,为评估HBoV3的流行传播特征和致病性提供了基础性数据。
二、北京地区健康人群中人博卡病毒的血清流行率调查
利用杆状病毒系统表达了HBoV1、2、3的病毒样颗粒,采用CsCl密度梯度离心纯化后,经电子显微镜和Western blot方法进行了鉴定。利用HBoV1、2、3的病毒样颗粒,建立了HBoV1、2、3 IgG抗体ELISA检测方法。在此基础上对642份北京地区健康人群的不同年龄组血清样本中HBoV1、2、3 IgG抗体的流行情况进行了检测,结果显示：人群中普遍感染HBoV1,在0.5-5岁的儿童,HBoV1抗体的阳性率大于HBoV2和HBoV3。HBoV的阳性率随年龄的增加而增高,20岁以后的成人HBoV1、2、3抗体阳性率均达到100%。虽然HBoV1、2、3抗体滴度随年龄的增加而增高,但HBoV2和HBoV3抗体滴度与HBoVl相比一直处于较低水平。这些结果提示不同型的HBoV流行水平存在差异,HBoVl是主要的HBoV的流行型。此外,建立了HBoV1 IgM抗体ELISA检测方法,具有良好的灵敏度、特异度及诊断符合率,可以用于HBoV1早期感染的血清学检测。
三、人博卡病毒VP2表位的筛选
制备HBoV1、2、3的VP2蛋白的鼠抗血清,和HBoV1、2、3的病毒样颗粒分别进行Western blot和ELISA检测,结果表明HBoV1、2、3之间存在血清学交叉反应。为了鉴定用于HBoV1、2、3血清学检测的特异性抗原,利用噬菌体载体构建了人博卡病毒VP2噬菌体肽库。分别用抗HBoV1、2、3的VP2蛋白的鼠抗血清和HBoV感染阳性的人血清对噬菌体肽库进行了生物淘洗,并使用ELISA方法筛选阳性肽,通过对阳性肽的基因测序及分析,初步获得了2个可能含有HBoV1、2、3的共有表位和1个HBoV3的特异性表位的区域。
综上所述,本研究首次发现了HBoV3核酸在急性呼吸道感染样本中的存在；通过血清流行病学调查,揭示了不同HBoV亚型的流行水平的差异,建立了HBoV早期感染的血清学诊断方法；首次评价了HBoV1、2、3之间的血清学交叉反应关系,并筛选出可能含有HBoV1、2、3的共有表位和HBoV3的特异性表位的区域,。本研究为深入揭示HBoV的流行特征和与致病性的关系以及研发免疫学检测技术提供了必要的研究基础。
New emerging infectious diseases damage human health seriously, affect social stabilization and threaten state security, which has earned global attention. Human bocavirus (HBoVs), firstly identified in 2005, has been reconized as a potential pathogen of respiratory infection. Since its first identification, four species of HBoVs (HBoV1-4) have been reported. However, the pathogenicity of HBoV species remains unclear. At present, mostly prevalence data were about HBoV1 and HBoV2, while the data about HBoV3 and HBoV4 were sparse. Prevalence of the four species HBoVs in the population has not been fully assessed. For the high amino acids homology between HBoV 1,2 and 3 species, serological cross-reactivities among the three species of HBoVs have also not been addressed, which reasied difficulties in the development and evaluation of the immunological assays for HBoVs.
In the present study, the prevalence of HBoV1-4 in children with acut lower respiratory tract infection (ALRTIs) and acute gastroenteritis (AGE) and the seroprevalence of HBoV1,-2 and-3 in healthy population were investigated. Moreover, the cross-reactivities between different HBoV species and poosible epitopes of HBoVs VP2 were investigated. The main result were displayed as follow:
1. Prevalence of HBoVl-4 in children suffered from ALRTIs and AGE in Beijing
Nasopharyngeal aspirates (NPAs) were collected from 1,238 hospitalized children with acute LRTIs at Beijing Children's Hospital between March 2008 and July 2010. These samples were assayed for HBoVs by a universal nested PCR. The results showed that of all the ALRTIs specimens tested,11.4% were positive for HBoVs, with 10.6% positive for HBoV1,0.4% for HBoV2 and 0.4% for HBoV3. No specimens were positive for HBoV4. Of patients with HBoVs,93.6% were≤5 years old. Similar to previous reports, other Additional respiratory pathogens were detected in 85.1% patients, The seasonal distribution of HBoVs is not obvious during the study period.
Stool specimens were collected from 366 outpatients with AGE at Beijing Children's Hospitral between March 2006 and November 2007 and the presence of HBoVs in these samples were also evaluated by the aforementioned method. The results showed that of all the stool specimens tested, the positive rate of HBoVs were 12%, the distribution ages of positive sample from 2 to 24 months of age (median age 9 months). Of those specimens,2.5% were positive for HBoV1,9.0% for HBoV2 and 0.5% for HBoV3, HBoV4 was not detected. Notably,79.5% of HBoVs-positive samples were co-detected with other viral pathogens. Analysis of the HBoV3 genome sequence indicates that HBoV3 may be a recombinant derived from HBoV1 and HBoV2 located in the upstream of NS1 and VP1/2 reading frame, or from HBoV1 and HBoV4.
2. Seroprovalence of HBoV1-3 in healthy population in Beijing
To evaluate the prevalence of HBoV in human population, we expressed virus-like particles (VLPs) of HBoV1,-2, and-3 species using High five cells and purified these VLPs by CsCl gradient centrifugation. After verification by electron microscopy and Western blot analysis, these VLPs were used as antigens for setting up ELISA methods to detect specific IgG antibodies against HBoV1, HBoV2 and HBoV3 in serum samples collected from 642 healthy people in different age groups respectively from Beijing. Ubiquitous infection of HBoV 1 was found in healthy population. In the children aged from 6 months to 5 years old, the seropositive rate of HBoV1 was higher than that of HBoV2 and HBoV3. The seropositive rates of HBoV1,-2, and-3 increased with age. In individuals over 20 years old, the seropositive rate of HBoV1,-2, and-3 reached almost 100%. In contrast, the antibodies titer of HBoV2, and-3 were not obviously boosted with age and kept at a relatively low level compared with HBoV1. Taken together, our data indicate a differential prevalence of HBoV species in human. Depite the prevalence of HBoV 1-3 were detected, HBoV1 may be dominant in the HBoV epidemic. Moreover, we also demonstrated that ELISA detection for HBoV1 IgM is effective for the early detection of HBoV infection.
3. Identification of epitopes in VP2 protein of HBoV1,2, and-3 using phage display peptide library
Serological cross-reactivities among HBoV1,-2,-3 were found by using HBoV VLPs react to murine sera against HBoV VP2 protein by Western blot and ELISA assays. To identify specific antigens for HBoVs serology diagnosis, an HBoV VP2 gene phage display random library was constructed. The HBoV VP2 gene phage peptide display library was selected by antisera from immunized mice with recombinant HBoV1,-2, and-3 VP2 protein and positive human sera. Positive clones were idedified by ELISA and further analyzed by gene sequencing. Two regions harboring possible epitopes that are conserved between HBoV1,-2, and-3 and one region harboring possible HBoV3 specific epitope were identified.
In conclusion, we report the first detection of HBoV3 DNA in the respiratory tract samples. Diffrential prevalence was found among different HBoVs by seroprevalence investigation. Several possible common epitope regions and one possible specific epitope region in HBoV1,2,3 were first identified. This study provides basis for evaluatintg the epidemic and pathogenesis and for developing immunological assays for HBoVs.
引文
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