本文選題：人博卡病毒2(HBoV2) + 嬰幼兒��； 參考：《蘭州大學(xué)》2011年博士論文

【摘要】：目的：腹瀉病是引起全世界兒童發(fā)病和死亡的主要原因之一,輪狀病毒、杯狀病毒、腺病毒和星狀病毒是嬰幼兒腹瀉的常見病毒性病原,但是仍然有相當(dāng)一部分的腹瀉病例檢測不到確切病原,對患兒的診斷及治療造成很大不便。人博卡病毒1-4型是近幾年新發(fā)現(xiàn)的細(xì)小病毒,且被推測可能與腹瀉有關(guān),但是由于缺乏大量病例對照的資料,人博卡病毒究竟是嬰幼兒腹瀉的致病原之一,或者僅僅是腸道的“過路者”,還沒有得到確切結(jié)論。本次研究擬通過病例對照研究來探討上述疑問。 方法：2006年7月至2008年6月,共收集蘭州大學(xué)第一醫(yī)院兒科632份住院腹瀉患兒和162份健康嬰幼兒的糞便標(biāo)本,采用ELISA、PCR、RT-PCR等方法檢測輪狀病毒,杯狀病毒,星狀病毒及腺病毒,對陽性標(biāo)本進(jìn)行基因測序。采用PCR方法檢測HBoV1-4型并進(jìn)行基因測序,同時通過實(shí)時熒光定量PCR方法確定HBoV2病毒載量。使用SPSS11.5進(jìn)行統(tǒng)計學(xué)分析,MEGA4.1軟件包進(jìn)行基因序列分析。 結(jié)果：病例組標(biāo)本中,輪狀病毒為最常見的病原,陽性率為45.3%,其次為杯狀病毒(10.1%),星狀病毒(4.9%),和腺病毒(4.7%)。HBoV1和]HBoV3的檢出率分別為27(4.3%),6(0.9%),HBoV4未檢出。HBoV2的檢出率高達(dá)20.4%(129/632),甚至超過了杯狀病毒,僅次于輪狀病毒。162份對照組中,輪狀病毒檢出3例,星狀病毒檢出1例,杯狀病毒和腺病毒各檢出1例,HBoV1檢出4例,HBoV3和HBoV4未檢出,HBoV2檢出率為12.3%(20/162).摒除年齡因素的影響后,多因素回歸分析結(jié)果顯示HBoV1和HBoV3與嬰幼兒腹瀉沒有疾病相關(guān)性,HBoV2與嬰幼兒腹瀉的疾病相關(guān)性(OR=1.269,CI=0.704-2.288)弱于輪狀病毒、杯狀病毒、腺病毒和星狀病毒。且病例組和對照組中HBoV2的核酸病毒載量均較低,高于104 copies/ml的高病毒載量標(biāo)本很少。 結(jié)論：本次研究首次在中國嬰幼兒腹瀉標(biāo)本中檢測到HBoV2和HBoV3,且HBoV1-3型的流行病學(xué)特征各不相同,病例組和對照組中檢出率有一定差異,通過統(tǒng)計學(xué)分析表明HBoV1和HBoV3與嬰幼兒腹瀉沒有疾病相關(guān)性,HBoV2盡管具有較高的檢出率,但是與嬰幼兒腹瀉的疾病相關(guān)性弱于常見腹瀉病毒。 目的：人博卡病毒2型(human bocavirus 2, HBoV2)是最近在兒童糞便標(biāo)本中發(fā)現(xiàn)的新的細(xì)小病毒,自被發(fā)現(xiàn)以后在糞便標(biāo)本中被頻繁檢出,并被認(rèn)為可能是急性腹瀉的致病原之一,但是在呼吸道標(biāo)本中很少檢出。迄今為止,所有檢測HBoV2的方法均是采用巢式PCR,即費(fèi)時費(fèi)力又容易出現(xiàn)假陽性,本次研究的目的即建立高靈敏度和特異度的實(shí)時熒光定量PCR (Real-time PCR)方法用于檢測HBoV2。 方法：2007年11月到2008年10月在山西太原市兒童醫(yī)院共收集了345份因腹瀉住院的嬰幼兒糞便標(biāo)本,根據(jù)HBoV2 NP1基因片段保守區(qū)設(shè)計了引物和熒光探針,并使用普通PCR擴(kuò)增了HBoV2 NP1基因中587bp長度片段并制備成陽性質(zhì)粒作為Real-time PCR的陽性標(biāo)準(zhǔn)品,按照倍比稀釋的方法稀釋10個濃度梯度評估Real-time PCR方法的敏感性。同時使用Kapoor等學(xué)者設(shè)計的常規(guī)巢式PCR方法進(jìn)行HBoV2檢測,與Real-time PCR方法進(jìn)行比較。 結(jié)果：對HBoV2 NP1質(zhì)粒不同濃度梯度(101-108拷貝)的DNA進(jìn)行線性擴(kuò)增,經(jīng)評估此方法的檢測低限為10個拷貝/mL。345份糞便標(biāo)本分別使用Real-time PCR和常規(guī)巢式PCR方法進(jìn)行HBoV2檢測,使用real-time PCR方法,85(24.6%,85/345)份糞便標(biāo)本為HBoV2陽性,平均病毒載量為1.02×104 copies/mL(從1.67×102 to 4.27×109 copies/mL)。使用常規(guī)巢式PCR進(jìn)行檢測,57(16.5%,57/345)份糞便標(biāo)本為PCR電泳鑒定陽性,PCR產(chǎn)物測序結(jié)果顯示其中52份為HBoV2,4份為HBoV1,1份為HBoV 3。19份標(biāo)本為常規(guī)巢式PCR單獨(dú)陽性,19份標(biāo)本根據(jù)所擴(kuò)增NS1片段測序結(jié)果,16份為HBoV2,16份HBoV2陽性標(biāo)本使用Real-timePCR重復(fù)擴(kuò)增3次,其中8份標(biāo)本擴(kuò)增陽性,均為其中一次陽性,且病毒載量均較低,均為10個拷貝以下(2.01×100-6.99×100 copies/mL)。 結(jié)論：本次研究中我們建立了一種Real-time PCR方法進(jìn)行HBoV2檢測。通過使用倍比稀釋的質(zhì)粒DNA評估此方法的敏感性、特異性及HBoV2 DNA擴(kuò)增的可靠性及可重復(fù)性,結(jié)果證明此定量擴(kuò)增方法完全可行,且敏感性、特異性等性能均高于常規(guī)巢式PCR方法,是較理想的熒光定量PCR方法,為以后的HBoV2研究提供了較方便和實(shí)用的新的檢測方法。 目的：人博卡病毒1-4型是近幾年新發(fā)現(xiàn)的病毒,之前的研究提出HBoV2不同亞型之間存在重組現(xiàn)象,HBoV3可能是HBoV1和]HBoV2的重組體,HBoV4基因組內(nèi)部也存在重組現(xiàn)象,但是由于基因序列有限及研究方法的局限性,HBoV1-4之間確切的進(jìn)化關(guān)系沒有得到證實(shí)。本次研究擬針對以上不足進(jìn)一步闡明HBoV1-4之間的進(jìn)化關(guān)系。 方法：在蘭州地區(qū)收集嬰幼兒腹瀉病例組和對照組糞便標(biāo)本,使用PCR方法檢測]HBoV1、HBoV2、HBoV3及HBoV4。使用特異引物擴(kuò)增HBoV2各基因片段序列,使用Genome Walking Kit擴(kuò)增HBoV2基因組末端序列并拼接出完整基因組序列。使用MEGA4.1等軟件整理基因序列并繪制進(jìn)化樹,按照序列同源性篩選出代表序列,使用RDP3等軟件檢測基因序列重組信號,并使用Shimodaira-Hasegawa test等方法對重組信號進(jìn)行驗(yàn)證。 結(jié)果：病例組與對照組的HBoV2 NS1片段基因序列彼此之間的同源性很高,基因進(jìn)化樹分析也.顯示兩組基因片段的拓?fù)浣Y(jié)構(gòu)特點(diǎn)具有較高的一致性。分析結(jié)果提示HBoV2不同毒株間存在重組位點(diǎn),然而通過Shimodaira和Hasegawa(SH)檢測,可以發(fā)現(xiàn)這些可能的breakpoints兩側(cè)的進(jìn)化樹拓?fù)浣Y(jié)構(gòu)并不是顯著地不一致。 分析結(jié)果顯示HBoV1, HBoV2, HBoV3和HBoV4基因組均存在一定的重組信號,但是HBoV3存在的重組信號最強(qiáng),其它3種病毒存在的重組信號較弱且沒有重要意義。HBoV3可能是HBoV1和HBoV4的重組體。然而,進(jìn)一步的分析發(fā)現(xiàn)HBoV3的VP1和VP2基因與HBoV2(?)同源性和與HBoV4的同源性相似。 結(jié)論：以上結(jié)果提示蘭州地區(qū)腹瀉人群與健康人群中流行的HBoV2來自同一毒株,且本研究提示HBoV2可以引起嬰幼兒無癥狀感染。盡管有證據(jù)支持HBoV2型內(nèi)部的重組,但是重組信號的可信度不強(qiáng),分析中出現(xiàn)的重組現(xiàn)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)的重組體。
[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).

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R373;R725.1

【共引文獻(xiàn)】

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