【作者】 赵扬；

【导师】 金玉； 段招军；

【作者基本信息】 兰州大学 ， 儿科学， 2010， 硕士

【摘要】 实验目的了解兰州地区急性呼吸道感染(ARTIs)患儿中呼吸道病原谱的分布现状,为本地区儿童急性呼吸道感染的防治提供基础数据。明确新发现病毒人博卡2病毒在呼吸道中的致病性。对象及方法1收集兰州大学第一医院儿科2008年11月到2009年10月因急性呼吸道感染就诊患儿的鼻咽抽吸物(Nasopharyngea aspirates, NPAs)和门诊患儿的咽拭子(throat swabs)及正常体检儿童的呼吸道分泌物标本。2分别采用聚合酶链反应(PCR)检测呼吸道合胞病毒(RSV)、流感病毒(IFVA、B型)、和副流感病毒(PIV 1-3型)、鼻病毒(HRV)、人偏肺病毒(HMPV)、冠状病毒-NL63(HCOV-NL63)、冠状病毒-HKU1(HCOV-HKU1)、冠状病毒-229E(HCOV-229E)、冠状病毒-OC43(HCOV-OC43)、多瘤病毒WU (WUPyV)和多瘤病毒KI (KIPyV) 14种常见及新发现呼吸道感染相关的病毒。结合详细的临床资料,做出临床流行病学分析。3建立博卡病毒的病例对照研究：采用巢式PCR (Nest-pcr)方法检测急性呼吸道感染患儿和正常儿童呼吸道标本中的HBoV-1和HBoV-2基因,并对扩增产物测序及进行Blast基因序列比对。讨论其在呼吸道中的致病性。结果1.在收集的510份呼吸道标本中,共检出病毒感染阳性病例382例(检出率74.9%)。其中RSV阳性扩增产物160份,检出率为31.4%；IFVA阳性扩增产物53份,检出率为10.4%; IFVB阳性扩增产物25份,检出率为4.9%；PIV3阳性扩增产物83份,检出率为16.1%；HRV阳性扩增产物48份,检出率为9.4%；HMPV阳性扩增产物16份,检出率为0.4%；HCoV-NL63阳性扩增产物3份,检出率为0.6%；HCoV-HKU1阳性扩增产物2份,检出率为0.4%; WUPyV阳性扩增产物7份,检出率为1.4%,KIPyV阳性扩增产物15份,检出率为2.9%,未检出PIV-1/-2和HCoV-229E/OC43。314例男性患者中病毒感染216例(58.3%),女性196例感染166例(55.5%),男女感染率的之比为1.3：1；患儿年龄最小10天,最大14岁,中位年龄28个月。其中1岁以下患儿病毒感染208例,占总病毒感染人数的54.5%；1～3岁患儿病毒感染84例,占22%；3～5岁患儿病毒感染54例,占14.1%；大于5岁患儿病毒感染36例,占9.4%。随着年龄的增长,病毒阳性检出率逐渐下降。本地区儿童病毒性急性呼吸道感染的季节分布较明显,以冬春季节为主。510份病例组呼吸道患儿标本检测到7例HBoV-2阳性扩增产物,检出率为1.4%。将阳性PCR产物进行序列测定及分析,发现其与GeneBank中已知标准株(Human bocavirus 2 strain HBoV2B-NI-213)序列的同源性在98%-100%之间,提示HBoV-2兰州株的核苷酸变异较小。患儿感染年龄从4个月到65个月,中位年龄8.5个月,除一例标本外,其余均在2岁以下儿童。全部为男性患儿。检出病例主要在冬春季节,12月检出最多。该病毒混合感染率高达57.1%,与RSV混合感染最多。临床诊断包括喘息性支气管肺炎、急性支气管肺炎、急性支气管炎,其中喘息性支气管肺炎最多见,为5例。HBOV-2在对照儿童中检出率为1.4%(4/290),HBOV-2阳性的患儿均≤6个月,标本分别在3月,5月,6月,8月中检出,男女比例为3：1。病例组中HBOV-1在病例组中检出率为14.3%(73/510),正常儿童中HBOV-1的检出率为3.1%(9/290)。结论1本研究中74.9%的患儿病毒检测阳性,结合本课题组2006-2008年的研究结果说明,病毒病原在兰州地区儿童呼吸道感染中仍占有重要地位。在2008-2009年的研究中发现儿童病毒性呼吸道感染病原以RSV、PIV-3、HRV及IFVA占主要地位,与2007-2008年相比IFVA及PIV-3两种呼吸道病毒在本地区的感染有所上升,跟2007-2008的呼吸道病毒感染谱略有差异,呼吸道病毒病原分布具有明显的季节分布特征,以冬春季节多见。2 HBOV-2与疾病：P>0.005 (Fisher’s Exact Test)无统计学意义。HBVO-2感染与呼吸道疾病无关。HBOV-1与疾病：p<0.005 (Pearson Chi-Square)有统计学意义,病例组中感染HBOV-1更高。HBOV-1可以引起呼吸道疾病。更多还原

【Abstract】 ObjectiveIn this study, in order to provide basic data and scientific evidence for the prevention and control of acute respiratory tract infection (ARTI), we investigated the common and potential viral etiological agent of ARTI among children in Lanzhou area. Meanwhile, we identified the pathogenesis of HBoV-2 in the respiratory tract infection.Subjects and Methods510 Nasopharyngea aspirates (NPAs) and throat swabs specimens were collected from children with acute respiratory infections and 290 specimes from healthy children from November 2008 to October 2009 in Lanzhou area of china.RSV, Influenza A and B, Parainflurnzal-3, HRV, HMPV, HCoV-NL63, HCoV-HKU1, HCoV-229E, HCoV-NL63, WUPyV and KIPyV were detected by RT-PCR and nest-PCR methods, and amplicons were identified by sequencing. Clinical and epidemiological analysis on acute respiratory tract infections among children which cause by diverse viruses were performed.The establishment of a case-control investigation:HBoV-1 and HBoV-2 were also tested by nested-PCR methods. The amplicons were identified by sequencing and the sequences were then used to search against those in the NCBI databases using Blast program. Meanwhile, The relatedness of HBoV-2 and newly identified Bocavirus to respiratory tract infection was also investigated in this study.Results1.382 samples were detected positive in 510 NPAs samples which were collected in 2008-2009 year and the total rate was 74.9%. Respectively, the rate of RSV was 31.4% (160/510), IFVA was 10.4%(53/510), IFVB was 4.9%(625/510), PIV3 was 16.1%(83/510), HRV was 9.4%(48/510), HMPV was 0.4%(16/510), HCoV-NL63 was 0.6%(3/510), HCoV-HKUl was 0.4%(2/510), WUPyV was 1.4%(7/510), KIPyV was 2.9%(15/510). PIV-1/-2 and HCoV-229E/OC43 were not detected. Among the 510 NPAs samples, the positive rate of samples collected from male patients was 58.3%(216/314) while that of samples from female cases was 55.5%(166/196). The ratio of male to female patients was 1.3:1. The median ages of the viruses infected patients was 28 months (range from 10 days to 14 years old).54.5%(208/510) children with viruses infection were under 1 year of age,18.1%(84/510) children were at age from 1 to 3 years,14.1%(54/510) children were at age from 3 to 5 years and 9.4%(36/510) children were older than 5 years old. The positive rate of virus detection decreased in accordance with the increasing of age. The detected rate had no significant difference among the age groups (P=0.989>0.05). There were remarkable seasonal distributions of viral respiratory tract infections in Lanzhou area, viral respiratory tract infections predominated in spring and winter.2. The detection rate of HBoV-2 was 1.4%(7/510). The positive amplicons were sequenced and then analyzed by using Blast program. The results showed that the sequences shared 98%～100% homology with Human bocavirus 2 strain HBOV2B-NI-213 in the NCBI nucleotide database except single insertions and mutations. It indicated that there was few diversity in the HBoV-2 Lanzhou isolates. The median ages of the viruses infected children was 8.5 months (range from 4 months to 65 months). All infected children except one were under 2 years old and interestingly all of them were male. The detected cases of viral respiratory tract infections were mainly during spring and winter, especially in December (3 cases). The cases of HBoV-2 usually coinfected with other viruses, mostly with RSV. Clinical symptoms included asthmatic bronchial pneumonia, acute bronchial pneumonia and acute bronchitis, especially asthmatic bronchial pneumonia (5 cases). The detection rate of HBOV-2 among the control children was 1.4%(4/290). The infected children of HBOV-2 were=6 months old. The samples detected positive were in March, May, June and August and the ratio of male to female were 3:1. The detection rate of HBOV-1 among the infected children was 14.3%(73/510) while it was 3.2% of healthy children.ConclusionsViral infection is the main reason of ALRTI of children in Lanzhou area and 74.9% of infected children were detected positive. RSV, PIV-3, HRV and IFVA are major causes of respiratory tract infections among children in this area. But there was a slightly different spectrum of respiratory tract infection representing the increasing of IFVA and PIV-3 infections comparing with that 2007-2008 year, and obvious seasonal distribution, especially in winter and spring.The relatedness of HBoV2 to respiratory tract infection:there was no significance in statistics (P>0.005, Fisher’s Exact Test). It indicated that HBoV-2 has no relationship with respiratory tract infection. Whereas, The HBoV-1 is related to respiratory tract infection according to analysis in statistics (p<0.005, Pearson Chi-Square). The detection rate of infected children was higher than that of control cases. And HBoV-1 can cause respiratory tract infection.更多还原