博卡病毒流行病学、致病性及基因进化研究

发布时间：2018-04-19 15:14

本文选题：人博卡病毒2(HBoV2) + 婴幼儿　；参考：《兰州大学》2011年博士论文

【摘要】：目的：腹泻病是引起全世界儿童发病和死亡的主要原因之一,轮状病毒、杯状病毒、腺病毒和星状病毒是婴幼儿腹泻的常见病毒性病原,但是仍然有相当一部分的腹泻病例检测不到确切病原,对患儿的诊断及治疗造成很大不便。人博卡病毒1-4型是近几年新发现的细小病毒,且被推测可能与腹泻有关,但是由于缺乏大量病例对照的资料,人博卡病毒究竟是婴幼儿腹泻的致病原之一,或者仅仅是肠道的“过路者”,还没有得到确切结论。本次研究拟通过病例对照研究来探讨上述疑问。方法：2006年7月至2008年6月,共收集兰州大学第一医院儿科632份住院腹泻患儿和162份健康婴幼儿的粪便标本,采用ELISA、PCR、RT-PCR等方法检测轮状病毒,杯状病毒,星状病毒及腺病毒,对阳性标本进行基因测序。采用PCR方法检测HBoV1-4型并进行基因测序,同时通过实时荧光定量PCR方法确定HBoV2病毒载量。使用SPSS11.5进行统计学分析,MEGA4.1软件包进行基因序列分析。结果：病例组标本中,轮状病毒为最常见的病原,阳性率为45.3%,其次为杯状病毒(10.1%),星状病毒(4.9%),和腺病毒(4.7%)。HBoV1和]HBoV3的检出率分别为27(4.3%),6(0.9%),HBoV4未检出。HBoV2的检出率高达20.4%(129/632),甚至超过了杯状病毒,仅次于轮状病毒。162份对照组中,轮状病毒检出3例,星状病毒检出1例,杯状病毒和腺病毒各检出1例,HBoV1检出4例,HBoV3和HBoV4未检出,HBoV2检出率为12.3%(20/162).摒除年龄因素的影响后,多因素回归分析结果显示HBoV1和HBoV3与婴幼儿腹泻没有疾病相关性,HBoV2与婴幼儿腹泻的疾病相关性(OR=1.269,CI=0.704-2.288)弱于轮状病毒、杯状病毒、腺病毒和星状病毒。且病例组和对照组中HBoV2的核酸病毒载量均较低,高于104 copies/ml的高病毒载量标本很少。结论：本次研究首次在中国婴幼儿腹泻标本中检测到HBoV2和HBoV3,且HBoV1-3型的流行病学特征各不相同,病例组和对照组中检出率有一定差异,通过统计学分析表明HBoV1和HBoV3与婴幼儿腹泻没有疾病相关性,HBoV2尽管具有较高的检出率,但是与婴幼儿腹泻的疾病相关性弱于常见腹泻病毒。目的：人博卡病毒2型(human bocavirus 2, HBoV2)是最近在儿童粪便标本中发现的新的细小病毒,自被发现以后在粪便标本中被频繁检出,并被认为可能是急性腹泻的致病原之一,但是在呼吸道标本中很少检出。迄今为止,所有检测HBoV2的方法均是采用巢式PCR,即费时费力又容易出现假阳性,本次研究的目的即建立高灵敏度和特异度的实时荧光定量PCR (Real-time PCR)方法用于检测HBoV2。方法：2007年11月到2008年10月在山西太原市儿童医院共收集了345份因腹泻住院的婴幼儿粪便标本,根据HBoV2 NP1基因片段保守区设计了引物和荧光探针,并使用普通PCR扩增了HBoV2 NP1基因中587bp长度片段并制备成阳性质粒作为Real-time PCR的阳性标准品,按照倍比稀释的方法稀释10个浓度梯度评估Real-time PCR方法的敏感性。同时使用Kapoor等学者设计的常规巢式PCR方法进行HBoV2检测,与Real-time PCR方法进行比较。结果：对HBoV2 NP1质粒不同浓度梯度(101-108拷贝)的DNA进行线性扩增,经评估此方法的检测低限为10个拷贝/mL。345份粪便标本分别使用Real-time PCR和常规巢式PCR方法进行HBoV2检测,使用real-time PCR方法,85(24.6%,85/345)份粪便标本为HBoV2阳性,平均病毒载量为1.02×104 copies/mL(从1.67×102 to 4.27×109 copies/mL)。使用常规巢式PCR进行检测,57(16.5%,57/345)份粪便标本为PCR电泳鉴定阳性,PCR产物测序结果显示其中52份为HBoV2,4份为HBoV1,1份为HBoV 3。19份标本为常规巢式PCR单独阳性,19份标本根据所扩增NS1片段测序结果,16份为HBoV2,16份HBoV2阳性标本使用Real-timePCR重复扩增3次,其中8份标本扩增阳性,均为其中一次阳性,且病毒载量均较低,均为10个拷贝以下(2.01×100-6.99×100 copies/mL)。结论：本次研究中我们建立了一种Real-time PCR方法进行HBoV2检测。通过使用倍比稀释的质粒DNA评估此方法的敏感性、特异性及HBoV2 DNA扩增的可靠性及可重复性,结果证明此定量扩增方法完全可行,且敏感性、特异性等性能均高于常规巢式PCR方法,是较理想的荧光定量PCR方法,为以后的HBoV2研究提供了较方便和实用的新的检测方法。目的：人博卡病毒1-4型是近几年新发现的病毒,之前的研究提出HBoV2不同亚型之间存在重组现象,HBoV3可能是HBoV1和]HBoV2的重组体,HBoV4基因组内部也存在重组现象,但是由于基因序列有限及研究方法的局限性,HBoV1-4之间确切的进化关系没有得到证实。本次研究拟针对以上不足进一步阐明HBoV1-4之间的进化关系。方法：在兰州地区收集婴幼儿腹泻病例组和对照组粪便标本,使用PCR方法检测]HBoV1、HBoV2、HBoV3及HBoV4。使用特异引物扩增HBoV2各基因片段序列,使用Genome Walking Kit扩增HBoV2基因组末端序列并拼接出完整基因组序列。使用MEGA4.1等软件整理基因序列并绘制进化树,按照序列同源性筛选出代表序列,使用RDP3等软件检测基因序列重组信号,并使用Shimodaira-Hasegawa test等方法对重组信号进行验证。结果：病例组与对照组的HBoV2 NS1片段基因序列彼此之间的同源性很高,基因进化树分析也.显示两组基因片段的拓扑结构特点具有较高的一致性。分析结果提示HBoV2不同毒株间存在重组位点,然而通过Shimodaira和Hasegawa(SH)检测,可以发现这些可能的breakpoints两侧的进化树拓扑结构并不是显著地不一致。分析结果显示HBoV1, HBoV2, HBoV3和HBoV4基因组均存在一定的重组信号,但是HBoV3存在的重组信号最强,其它3种病毒存在的重组信号较弱且没有重要意义。HBoV3可能是HBoV1和HBoV4的重组体。然而,进一步的分析发现HBoV3的VP1和VP2基因与HBoV2(?)同源性和与HBoV4的同源性相似。结论：以上结果提示兰州地区腹泻人群与健康人群中流行的HBoV2来自同一毒株,且本研究提示HBoV2可以引起婴幼儿无症状感染。尽管有证据支持HBoV2型内部的重组,但是重组信号的可信度不强,分析中出现的重组现象可能是由于其它原因引起的；HBoV3可能是HBoV和(HBoV2、HBoV4父系序列)的重组体。
[Abstract]:Objective: diarrhoea is one of the main causes of disease and death in children all over the world. Rotavirus, goblet virus, adenovirus and stellate virus are common viral pathogens of infantile diarrhea. However, a considerable number of diarrhoea cases are still not detected, and the diagnosis and treatment of children are very inconvenient. Human Boka Virus 1-4 is a newly discovered parvovirus in recent years and is presumed to be associated with diarrhea. However, due to the lack of a large number of case control data, human Boka virus is one of the pathogenic factors of infantile diarrhea, or only the "passerby" in the intestinal tract. The study is to be studied by case control study. Discuss the above questions.
Methods: from July 2006 to June 2008, 632 stool specimens from children with diarrhea and 162 healthy infants in First Hospital Affiliated to Lanzhou University were collected. Rotavirus, goblet virus, stellate virus and adenovirus were detected by ELISA, PCR and RT-PCR, and the positive specimens were sequenced by PCR method. The HBoV1-4 type was detected by PCR method. The gene sequence was sequenced and the HBoV2 virus load was determined by real time fluorescence quantitative PCR. SPSS11.5 was used for statistical analysis, and the MEGA4.1 software package was used to carry out the gene sequence analysis.
Results: Rotavirus was the most common pathogen in the case group, the positive rate was 45.3%, followed by the goblet virus (10.1%), the stellate virus (4.9%), and the detection rates of adenovirus (4.7%).HBoV1 and]HBoV3 were 27 (4.3%), 6 (0.9%), and HBoV4 was not detected by 20.4% (129/632), even more than the goblet virus, second only to rotavirus. In the control group, 3 cases were detected by rotavirus, 1 were stellate virus detection, 1 cases were detected by goblet virus and adenovirus, 4 cases were detected by HBoV1, HBoV3 and HBoV4 were not detected, and the detection rate of HBoV2 was 12.3% (20/162). After eliminating the influence of age factors, the results of multivariate regression analysis showed that HBoV1 and HBoV3 had no disease correlation with infantile diarrhea, HBoV2 The association with infantile diarrhea (OR=1.269, CI=0.704-2.288) is weaker than rotavirus, goblet virus, adenovirus and stellate virus. The HBoV2 virus load of HBoV2 in case group and control group is low, and the high viral load samples higher than that of 104 copies/ml are very few.
Conclusion: HBoV2 and HBoV3 were detected for the first time in Chinese infantile diarrhea specimens, and the epidemiological characteristics of HBoV1-3 type were different. There was a certain difference in the detection rate between the case group and the control group. The statistical analysis showed that there was no correlation between HBoV1 and HBoV3 with infantile diarrhea, but HBoV2 had a high detection rate, but it had a higher detection rate. It is associated with diarrhea in infants and young children than diarrhea virus.
Objective: the human Boka virus 2 (human bocavirus 2, HBoV2) is a new parvovirus found in children's feces specimens recently. It has been found frequently in fecal specimens and is considered to be one of the pathogeny of acute diarrhea, but is rarely detected in respiratory specimens. So far, all methods for detecting HBoV2 The purpose of this study is to establish high sensitivity and specificity real-time fluorescence quantitative PCR (Real-time PCR) method for detecting HBoV2., which is the use of nested PCR, which is time-consuming and easy to be false positive.
Methods: 345 infants' feces were collected from November 2007 to October 2008 in Taiyuan children's Hospital, Shanxi. The primers and fluorescent probes were designed according to the conservative region of HBoV2 NP1 gene fragment. The 587bp length segment of the HBoV2 NP1 gene was amplified by common PCR and the positive plasmid was prepared as Real-time PCR. The positive standard was diluted by 10 concentration gradients to evaluate the sensitivity of the Real-time PCR method. At the same time, the conventional nested PCR method, which was designed by scholars such as Kapoor and other scholars, was used for HBoV2 detection and compared with the Real-time PCR method.
Results: the DNA of HBoV2 NP1 plasmids with different concentration gradient (101-108 copies) was amplified linearly. After evaluation, the detection limit of the method was 10 copies of /mL.345 feces samples using Real-time PCR and conventional nested PCR method for HBoV2 detection. Real-time PCR method was used, and 85 (24.6%, 85/345) fecal specimens were HBoV2 positive, average. The viral load was 1.02 * 104 copies/mL (from 1.67 * 102 to 4.27 x 109 copies/mL). The routine nested PCR was used to detect and 57 (16.5%, 57/345) fecal specimens were identified by PCR electrophoresis. The PCR product sequencing results showed that 52 copies of HBoV2,4 were HBoV1,1 shares of HBoV 3.19 samples as conventional nested PCR positive, 19 samples were based on The amplified NS1 fragment sequencing results showed that 16 samples of HBoV2,16 HBoV2 positive specimens were repeated 3 times with Real-timePCR, of which 8 specimens were positive, both of which were positive, and the viral load was low, all of which were below 10 copies (2.01 x 100-6.99 x 100 copies/mL).
Conclusion: in this study, we established a Real-time PCR method for HBoV2 detection. The sensitivity, specificity and reliability and repeatability of HBoV2 DNA amplification were evaluated by using the double dilution plasmid DNA. The results showed that the quantitative amplification method was completely feasible, and the sensitivity and specificity of the method were higher than that of the conventional nested method. The PCR method is an ideal fluorescence quantitative PCR method, which provides a more convenient and practical new detection method for HBoV2 research in the future.
Objective: human Boka virus type 1-4 is a newly discovered virus in recent years. Previous studies suggest that there is a recombination phenomenon between different subtypes of HBoV2. HBoV3 may be a recombinant of HBoV1 and]HBoV2, and there is a recombination phenomenon within the HBoV4 genome. However, the exact evolutionary relationship between HBoV1-4 and the limited genetic sequence and the limitations of the research methods This study is intended to further clarify the evolutionary relationship between HBoV1-4.
Methods: the fecal specimens of infantile diarrhea case group and control group were collected in Lanzhou area.]HBoV1, HBoV2, HBoV3 and HBoV4. were used to amplify the sequence of HBoV2 gene fragments using PCR method. Genome Walking Kit amplified HBoV2 genome terminal sequence and spliced the complete genome sequence with Genome Walking Kit. The sequence was plotted and the representative sequence was screened according to the sequence homology. The recombinant signal of gene sequence was detected by RDP3 and other software, and the recombinant signal was verified by Shimodaira-Hasegawa test.
Results: the homology of HBoV2 NS1 fragment sequences between the case group and the control group was high, and the gene evolution tree analysis also showed that the topological structure of the two sets of gene fragments had high consistency. The results suggested that the recombinant loci existed among the different strains of HBoV2, but the detection of Shimodaira and Hasegawa (SH) could be found. These possible evolutionary tree topologies on both sides of breakpoints are not significantly inconsistent.
The results showed that there were some recombinant signals in the HBoV1, HBoV2, HBoV3 and HBoV4 genome, but the recombinant signal of HBoV3 was the strongest. The other 3 viruses had weak recombination signals and no significant meaning.HBoV3 might be the recombinant of HBoV1 and HBoV4. However, further analysis found that VP1 and VP2 genes of HBoV3 are the same as HBoV2 (?). The origin is similar to the homology of HBoV4.
Conclusion: the above results suggest that the prevalence of HBoV2 in the diarrhea and healthy population of Lanzhou is from the same strain, and this study suggests that HBoV2 can cause asymptomatic infantile infantile infection. Although there is evidence to support the internal recombinant of HBoV2, the reliability of the recombinant signal is not strong, and the recombination phenomenon in the analysis may be due to other sources. HBoV3 may be the recombinants of HBoV and (HBoV2, HBoV4 paternal sequences).

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373;R725.1

【共引文献】