摘要
自2005年瑞典科学家Allander等人首次在呼吸道感染患儿的鼻咽吸出物中发现人博卡病毒(Human bocavirus, HBoV)以来,又陆续发现了3个新的HBoV基因型别—HBoV2～4。这些病毒属于细小病毒科(Parvoridae)细小病毒亚科(Parvovirinae)的博卡病毒属(Bocavirus),是迄今发现的继细小病毒B19之后能感染人的第二类细小病毒。HBoV在世界各地均有报道,许多国家已经报道了在不同标本中(如呼吸道标本、粪便、血清等)检测出HBoVs,旦绝大部分的研究是针对HBoV1,对HBoV2～4的研究报道还很有限。从现有的资料来看,越来越多的临床证据支持HBoVl为下呼吸道感染的致病病毒,而HBoV2～4主要在腹泻患儿的粪便标本中检出,这些感染肠道的HBoVs尤其是HBoV2在粪便标本中的检出率要高于其它基因型别,提示它们可能与儿童的急性胃肠炎有关。目前为止,HBoV尚不能体外培养,也无动物模型建立,阻碍了其复制机制及致病机理的研究。为揭示其复制机制,有研究在临床标本中检测了HBoV1、3的头-尾DNA序列和环状游离体结构,并在此基础上成功构建了HBoV1的感染性克隆,以绕开上述瓶颈进行致病机理的研究。鉴于腹泻病在我国儿童疾病谱中的重要地位,本研究对HBoV2进行了初步研究,以明确其与小儿腹泻病的可能相关性；得到了HBoV2包括头-尾序列在内的完整基因组,这是国际上的首次报道；并尝试构建HBoV2的感染性克隆,为阐明这一病毒的复制模式和致病机制打下基础。
首先,为了了解北京地区急性腹泻患儿中HBoV2的流行情况并了解这一病毒的基因组特征,收集了2010年11月至2012年10月到首都儿科研究所附属儿童医院门诊就诊的急性腹泻患儿的粪便标本1068例,应用针对HBoV2NS1基因的PCR引物进行荧光实时PCR检测。选择2例病毒载量较高的阳性标本进行HBoV2各基因片段的扩增并测序。将所测到的序列进行拼接后得到接近完整的HBoV2基因组序列并与GenBank中的相关序列进行比较分析。结果显示,所检测的2010年11月至2012年10月期间的1068例粪便标本中有24例HBoV2DNA检测阳性,阳性检出率为2.2%；各年龄组中,3-6月龄患儿中的HBoV2DNA阳性检出率最高,达3.6%(9/252),其次为6-9月龄(2.5%,5/203)的患儿,小于3月龄和大于3岁(含3岁)的患儿中未检测到HBoV2阳性标本；所检第一年度中,HBoV2日性检出率在7月最高,为7.0%(3/43),其次为9月(4.8%,2/41)和6月(4.8%,2/42),1-4月无阳性标本检出。第二年度中,HBoV2阳性检出率在9月最高,为4.5%(2/44),其次为7月(4.5%,2/45),11、12月及1-5月无阳性标本检出。24例HBoV2检测阳性的患儿除1例患儿年龄大于2岁以外,其余均为2岁以下儿童。其中5例患儿同时合并有诺如病毒感染、5例合并有轮状病毒感染、1例合并有腺病毒感染。经测序得到两株接近完整的HBoV2基因组序列BJQ19和BJQ390,长度分别为5273bp和5275bp；序列分析显示这两株序列的同源性为99.2%,与GenBank中来自中国上海的FJ375129株同源性最高,分别为99.1%和99.2%,为典型的HBoV2。上述结果表明,北京地区部分儿童的急性腹泻可能与HBoV2感染相关,且HBoV2感染在低年龄组腹泻儿童中更为常见。
其次,为了明确HBoV2基因组是否存在头-尾相接的环状游离体结构,进一步对已确证为HBoV2(?)日性的24例急性腹泻患儿的粪便标本进行了头尾序列的测定。第一年度,通过巢式PCR检测发现2例标本有预期目的条带,经测序证实其中1例(BJQ435)为HBoV2头-尾序列阳性标本,随后对各个片段进行扩增和测序得到了HBoV2完整的环状基因组,标记为HBoV2-C1(GenBank序列号为JX257046)。第二年度,检出2例HBoV2头-尾序列阳性标本(Q1032和Q1089)并经测序证实。HBoV2-C1全长含5307个核苷酸,含4个开放读码框(Open Reading Frame, ORF)依次含1923、648、2004、1617个核苷酸,分别编码NS1、NP1、VP1(?)口VP2蛋白。基因组两端即在VP1/VP2和NS1基因之间有520个核苷酸长度的非编码区(Non-coding region, NCR),在HBoV2-C1末端连接头尾的序列中有约50个核苷酸是在以往报告的HBoV末端序列中未发现过的,是本研究的新发现。对BoV2-C1和HBoV3-E1(JN086998)的二级结构预测表明,与HBoV3-E1相比,HBoV2-C1的“发卡结构2”更为复杂,其带有一个额外的兔耳样结构；另外一个主要的不同是,HBoV2-C15'末端结构中的茎环结构比文献报道的HBoV3-E1的分布更为分散。上述结果表明HBoV2基因组存在含头-尾序列的环状结构,这一发现对于这一病毒的复制机制补充了新的资料。
最后,在得到HBoV2环状基因组的基础上,进行了HBoV2全基因组重组质粒的构建。构建策略如下：①将基因组插入到适当的载体中,利用限制性内切酶将基因组切割下来,经凝胶电泳纯化后在体外使之自我环化,获得环状基因组。②设计含合适酶切位点的引物,利用扩增片段两端的酶切位点和载体自身的酶切位点,将其基因组分段依次插入到同一载体。将整个基因组分为4个片段进行扩增,其中采用重叠延伸PCR扩增得到了含头-尾序列的第四个片段HBoV2-4,并将各个片段插入到pGEM-T Easy载体得到了各个片段的重组质粒,然后通过酶切、连接反应及重叠延伸PCR扩增的方法将其余3个片段(HBoV2-1, HBoV2-2, HBoV2-3)拼接在一起,得到了含HBoV2-1+2+3片段的重组质粒。HBoV2-1+2+3和HBoV2-4经测序证实为HBoV2序列。全基因组的重组质粒仍在构建当中。
综上所述,本研究进行了北京地区急性腹泻患儿中HBoV2的流行情况的初步调查并对这一病毒的基因组特征进行了分析；完成了HBoV2完整的基因组序列的测定分析,报道了HBoV2基因组存在头-尾相接的环状结构,这是国际上的首次报道；并在此基础上成功构建了HBoV2-1+2+3片段和HBoV2-4片段的重组质粒。上述研究结果为进一步深入研究HBoV2与儿童腹泻的关系及其致病机理打下了基础。
Since human bocavirus (HBoV) was first discovered in nasopharyngeal aspirates from children with respiratory tract infections by Allander et al. in2005, three additional genotypes of HBoV have been reported in succession:HBoV2, HBoV3, and HBoV4. These HBoVs were classified into the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae) and considered to be the second parvovirus species discovered to date, besides parvovirus B19, which can cause infectious diseases in humans. HBoVs have been detected worldwide in different specimens (such as respiratory aspirates, stool, serum, etc.), but most studies were about HBoV1, and reports for HBoV2～4were still very limited. Accumulating clinical data in recent years have indicated that HBoV1is an important pathogen in lower respiratory tract illnesses, while HBoVs2～4are mainly detected in stool samples. The higher detection rate of HBoV2than other HBoV genotypes in stool specimens suggest that HBoV2may associate with acute gastroenteritis. To date, HBoV cannot be obtained by large-scale culture in vitro and there is no animal model for HBoVs, which impede the studies for the replication mechanism and pathogenic mechanism of HBoVs. To reveal its replication mechanism, head-to-tail sequences of HBoV1and episomal circular form of HBoV3genome have been detected in clinical specimens, and the HBoVl infectious clone have been successfully constructed to bypass the bottleneck for studying the pathogenic mechanism of HBoVs. Because of the importance of diarrheal diseases in the spectrum of diseases for children in our country, the prevalence of HBoV2was investigated in this study to find out the possible relevance between HBoV2and diarrheal diseases; a complete genome of HBoV2containing head-to-tail sequences was obtained, which is the first report in the world. This preliminary data is helpful for clarifying the replication model and pathogenesis of this virus in future.
The first step for investigating this emerging assumed gastroenteritis related virus was to detect the prevalence of HBoV2in pediatric patients with acute diarrhea in Beijing and the characteristics of the genome of the virus. To do that,1068stool specimens were collected from pediatric outpatients with acute diarrhea in Affiliated Children's Hospital of Capital Institute of Pediatrics during Nov.2010to Oct.2012. TaqMan-based Real-time polymerase chain reaction (PCR) was performed to detect HBoV2DNA from these specimens using primers targeting NS1gene. Two samples with high viral loads were selected from positive specimens for segmented amplification, and then the amplified fragments were cloned into the pGEM-T Easy vector, transformed into Escherichia coli DH5a and sequenced. Then genomic sequences assembled from those DNA fragments were compared with other parvovirus genomic sequences in the GenBank. From Nov.2010to Oct.2012, among1068specimens tested,24(2.2%) were HBoV2DNA positive.The highest positive rate was shown in infants in3-6month age group (3.6%,9/252) among different age groups followed by6-9month age group (2.5%,5/203). There were no positive specimens detected in the age groups of younger than3months and older than or equal to3years. In the first investigating year, the highest positive rate was shown in July (7%,3/43) through the whole year followed by September (4.8%,2/41), and June (4.8%,2/42). There was no positive specimen detected from January to April. In the second investigating year, the highest positive rate was shown in September (4.5%,2/44) through the whole year followed by July (4.5%,2/45). There was no positive specimen detected from Nov.2011to May.2012. All these24specimens positive for HBoV2DNA were collected from patients younger than2years old except one, who was older than2years old. Eleven of these24positive specimens were positive simultaneously for other gastroenteritis related viruses detected, including5for norovirus,5for rotavirus and1for adenovirus. Two almost complete HBoV2genomic sequences were assembled by sequence analysis for gene fragments amplified from specimens BJQ19and BJQ390, which were typical HBoV2, and were5273bp and5275bp in lengnth, respectively. They shared high homology with each other (99.2%), and shared the highest homology with FJ375129from Shanghai China (99.1%and99.2%) among other parvoviruses. These data suggest that some of acute diarrhea in pediatric patients in Beijing were associated with HBoV2, and infants and young children aged from3months to2years, were more likely to be infected by HBoV2.
The second step of the investigating approach was to determine if human bocavirus2(HBoV2) has a circular genome similar to the head-to-tail sequence of HBo V1and the episomal form of HBoV3. All of24HBoV2positive samples identified from children with acute diarrhea were further tested for a head-to-tail sequence. In the first year,2samples with expected PCR products were identified by nested PCR and one of them (BJQ435) was confirmed as positive for the head-to-tail sequence of HBoV2by sequencing. The complete circular genome of HBoV2was finally obtained by segmented amplification and sequencing, which was labeled as HBoV2-C1(GenBank No. JX257046). In the second year,2positive samples with head-to-tail sequence were detected by nested PCR and confirmed by sequencing. HBoV2-C1(BJQ435) was5307nt in length and was flanked with a520nt-long terminal non-coding region (NCR) between the VP1/VP2and NS1genes. There were four open reading frames (ORFs) in HBoV2-C1, which consisted of1923,648,2004and1617nt encoding NS1、NP1、VP1and VP2proteins respectively and50nucleotides linking the head to tail in HBoV2-C1, which was detected first time in this study. Prediction for the secondary structures of HBoV2-C1and HBoV3-E1showed that compared to HBoV3-E1, HBoV2-C1had a more complicated hairpin-2with an additional rabbit ear-like structure. The second major difference was that the stems and loops in the5'terminus structure were more scattered in HBoV2-C1than those in HBoV3-El. Our study indicates that the HBoV genome exists in the form of a head-to-tail monomer and provides more information for understanding the HBoV replication mechanism.
Finally, two strategies were used to construct a recombinant plasmid containing the complete genome of HBoV2based on the obtained HBoV2-C1genome:first, the genome is inserted into a suitable vector and then cut down by restriction endonuclease followed by purification with agarose gel electrophoresis. The circular genome is finally recovered in vitro by self-cyclization; Second, the segments of genome are inserted into the same vector in proper sequence using the restriction sites of the vector itself and the same restriction sites at both ends of the amplified fragments, which are generated by PCR using designed primers containing the appropriate restriction enzyme sites. The entire genome was divided into four fragments for amplification, of which the HBoV2-4fragment containing the head-to-tail sequence was obtained by overlap extension PCR. The other three fragments (HBoV2-1, HBoV2-2, and HBoV2-3) were ligated to produce fragment HBoV2-1+2+3by restriction enzyme digestion, ligation reactions and overlap extension PCR. Then, each fragment was inserted into the pGEM-T Easy vector to obtain recombinant plasmids. HBoV2-1+2+3and HBoV2-4were confirmed as HBoV2by sequencing. The recombinant plasmids containing the complete genome of HBoV2are still on going.
In summary, the prevalence of HBoV2in children with acute diarrhea in Beijing was investigated and the characteristics of the genome of this virus was analyzed in this study; complete HBoV2genome sequencing was accomplished identifying HBoV2has a circular genome with head-to-tail sequences for the first time in the world, and the recombinant plasmids containing HBoV2-1+2+3and HBoV2-4were successfully constructed. The data mentioned above will promote an intensive study for the relevance between HBoV2and pediatric diarrhea and the pathogenic mechanism of HBoV2in future.
引文
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