【摘要】：研究背景新發(fā)傳染病始終威脅著人類的健康,發(fā)現(xiàn)和鑒定新病毒以及確定新病毒與疾病的關(guān)系是預(yù)防、診斷和治療新發(fā)病毒性傳染病的首要任務(wù)。近十多年來,隨著生物技術(shù)的發(fā)展,越來越多的病毒被發(fā)現(xiàn),如冠狀病毒NL63、HKU1、MERS病毒、埃博拉病毒、博卡病毒和偏肺病毒等。盡管近年來對(duì)疾病病毒感染的病原學(xué)已有較深入的研究,但目前仍有一部分的疾病病因不明,部分未知的疾病病因可能是由未被發(fā)現(xiàn)的病毒所引起。WU多瘤病毒是2007年5月美國(guó)華盛頓大學(xué)醫(yī)學(xué)院科學(xué)家Gaynor通過高通量序列測(cè)定,從一名患有肺炎的3歲兒童的鼻咽抽吸物標(biāo)本中發(fā)現(xiàn)的一種新病毒。Saffold心病毒于1981年從一個(gè)8個(gè)月大的不明原因發(fā)熱病人的糞便中首次分離。在2007年,通過非序列依賴的單引物擴(kuò)增,確定為心病毒屬中感染人類的新種。研究報(bào)道,在患者的鼻咽抽吸物、糞便、血清等標(biāo)本中可以檢測(cè)出WU多瘤病毒和Saffold心病毒,因此可能是引起疾病的病原體之一。由于WU多瘤病毒和Saffold心病毒是新發(fā)現(xiàn)的病毒,近年來越來越引起廣泛的關(guān)注。當(dāng)前國(guó)內(nèi)對(duì)于這兩種病毒在中國(guó)地區(qū)的流行規(guī)律,致病性和基因特征缺乏了解,大多數(shù)的研究是僅僅局限在一種疾病中檢測(cè)分析這兩種病毒,缺乏對(duì)于病毒的流行規(guī)律、臨床特征和基因特征進(jìn)行分析。目的本研究通過對(duì)重慶醫(yī)科大學(xué)附屬兒童醫(yī)院的呼吸道感染患者鼻咽抽吸物標(biāo)本、急性腹瀉患者和手足口病患者的糞便標(biāo)本進(jìn)行病原體檢測(cè),對(duì)新發(fā)現(xiàn)的WU多瘤病毒、Saffold心病毒在兒童患者中的流行規(guī)律和基因進(jìn)化特征進(jìn)行系統(tǒng)性的分析,從而為下一步的病毒感染治療、疫情控制提供科學(xué)依據(jù)和參考意見。方法以重慶醫(yī)科大學(xué)附屬兒童醫(yī)院作為哨點(diǎn)監(jiān)測(cè)醫(yī)院,收集2012年1月到2015年12月的呼吸道感染患者的鼻咽抽吸物標(biāo)本、急性腹瀉患者的糞便標(biāo)本和手足口病患者的糞便標(biāo)本。通過采用PCR,RT-PCR,Real-Time PCR,Real-Time RT-PCR等方法檢測(cè)不同標(biāo)本中的病毒。呼吸道感染患者鼻咽抽吸物標(biāo)本檢測(cè)的常規(guī)病毒包括人腺病毒(human adenovirus,HAd V)、流感病毒(influenza virus,Flu)、人鼻病毒(human rhinovirus,HRV/HEV)、呼吸道合胞病毒(respiratory syncytial virus,RSV)、偏肺病毒(metapneumovirus,MPV)、副流感病毒(parainfluenza,PIV)、人博卡病毒(human bocavirus,HBo V)和人冠狀病毒(human coronavirus,HCo V)。急性腹瀉患者糞便標(biāo)本檢測(cè)的常規(guī)病毒包括輪狀病毒(rotavirus,Rt V)、諾如病毒(norovirus,No V)、腺病毒(adenovirus,Ad V)、扎如病毒(sapovirus,Sp V)和星狀病毒(astrovirus,At V)。手足口病患者糞便標(biāo)本中檢測(cè)的常規(guī)病毒是腸道病毒(Enterovirus,EV)、腸道病毒71型(Enterovirus 71,EV71)和柯薩奇病毒A16型(Coxsackievirus A16,CVA16)。呼吸道感染患者鼻咽抽吸物標(biāo)本、急性腹瀉患者糞便標(biāo)本和手足口病患者糞便標(biāo)本均檢測(cè)WU多瘤病毒(WU Polyomavirus,WUPy V)和Saffold心病毒(Saffold Cardiovirus,SAFV)。對(duì)WU多瘤病毒陽(yáng)性的標(biāo)本進(jìn)行全基因組擴(kuò)增,Saffold心病毒陽(yáng)性的樣本進(jìn)行VP1片段擴(kuò)增并分型,并進(jìn)行系統(tǒng)發(fā)育分析。使用Epidata3.1錄入數(shù)據(jù),采用SPSS 20.0軟件進(jìn)行統(tǒng)計(jì)分析,檢驗(yàn)水準(zhǔn)α=0.05。構(gòu)建系統(tǒng)發(fā)育進(jìn)化樹采用軟件Mega 7.0,使用maximum likelihood法,bootstrap值設(shè)定為1000進(jìn)行構(gòu)建和檢驗(yàn)。選擇壓力分析采用FEL、IFEL、MEME、SLAC四種方法篩選,同一位點(diǎn)至少在三種方法中檢出則判定為正選擇位點(diǎn),使用Data Monkey網(wǎng)站提交序列進(jìn)行分析(http://www.datamonkey.org/)。結(jié)果1.WU多瘤病毒在兒童患者中的流行規(guī)律與基因特征研究(1)本研究共檢測(cè)出WUPy V陽(yáng)性170例,總檢出率為4.7%。其中呼吸道感染患者鼻咽抽吸物標(biāo)本中WUPy V陽(yáng)性127例(7.8%),急性腹瀉患者糞便標(biāo)本中WUPy V陽(yáng)性25例(2.4%),手足口病患者的糞便標(biāo)本中WUPy V陽(yáng)性18例(1.9%),三者的陽(yáng)性檢出率之間差異具有統(tǒng)計(jì)學(xué)意義(P0.001),呼吸道感染患者WUPy V陽(yáng)性率最高。(2)在三種類型的病例中,WUPy V復(fù)合感染率為79.4%(135/170),合并一種病毒感染97例,合并兩種病毒感染31例,合并三種病毒及以上感染7例,單獨(dú)感染W(wǎng)UPy V的有35例。呼吸道感染患者復(fù)合感染率為79.5%(101/127),復(fù)合感染發(fā)生最多的病毒是HRV/HEV(28例);急性腹瀉患者復(fù)合感染率為76.0%(19/25),復(fù)合感染發(fā)生最多的病毒是Rt V(11例);手足口病患者復(fù)合感染率為83.4%(15/18),復(fù)合感染發(fā)生最多的病毒是EV71(9例)。(3)從檢測(cè)陽(yáng)性率的時(shí)間分布看,呼吸道感染患者在2013年5-6月WUPy V陽(yáng)性檢出率最高,急性腹瀉患者和手足口病患者在2013年7月出現(xiàn)一次檢測(cè)陽(yáng)性率高峰,提示在這一時(shí)間段,可能出現(xiàn)WUPy V感染的暴發(fā)。(4)從臨床癥狀上看,在呼吸道感染患者中,WUPy V復(fù)合感染的患者出現(xiàn)咳嗽,干Up音,濕Up音等癥狀的頻率大于WUPy V單獨(dú)感染的患者(P0.05)。在上呼吸道感染患者中WUPy V感染率為11.4%,且和非上呼吸道感染患者相比,差異具有統(tǒng)計(jì)學(xué)意義(P=0.018,OR=1.770,95%CI:1.103-2.842),肺炎患者中WUPy V感染率為7.8%。(5)本研究成功擴(kuò)增57株WUPy V全基因組序列,同源性在98.7%-100%。同Gen Bank上已經(jīng)上傳的其他地區(qū)的序列做系統(tǒng)進(jìn)化分析,顯示形成了I、II、III三個(gè)分支。本次研究中的序列聚集在Ia、Ic和IIIc,并且形成了一個(gè)新的亞支IIIc。來自呼吸道感染患者的35株序列,分別聚集于Ia(20株),Ic(8株),IIIc(7株)。來自急性腹瀉患者標(biāo)本中的12株序列,全部分布在Ia分支。來自手足口病患者標(biāo)本的10株序列,8株在Ia,2株在IIIc。(6)WUPy V選擇壓力分析顯示VP1片段的第82號(hào)位點(diǎn)為正選擇位點(diǎn),VP2,VP3,STAg,LTAg均未發(fā)現(xiàn)正選擇位點(diǎn)。2.Saffold心病毒在兒童患者中的流行規(guī)律與基因特征研究(1)本研究中共檢測(cè)出SAFV陽(yáng)性190例,總檢出率為2.0%。其中在呼吸道感染患者鼻咽抽吸物標(biāo)本中檢測(cè)出44例(1.3%),急性腹瀉患者糞便標(biāo)本中檢測(cè)出28例(0.9%),手足口病患者的糞便中檢測(cè)出118例(3.5%),三者的陽(yáng)性檢出率不同,差異具有統(tǒng)計(jì)學(xué)意義(P0.001),手足口病患者SAFV陽(yáng)性率最高。(2)SAFV總的復(fù)合感染率為73.2%(139/190),合并一種病毒感染116例,合并兩種病毒感染18例,合并三種病毒及以上感染5例,SAFV單獨(dú)感染51例。呼吸道感染患者復(fù)合感染率為84.1%(37/44),復(fù)合感染發(fā)生最多的病毒是RSV(12例);急性腹瀉患者復(fù)合感染率為67.9%(19/28),復(fù)合感染發(fā)生最多的病毒是Rt V(16例);手足口病患者復(fù)合感染率為70.3%(83/118),復(fù)合感染發(fā)生最多的病毒是EV71(36例)。(3)呼吸道感染患者中,大于36個(gè)月年齡組患者的SAFV陽(yáng)性率高于1-6個(gè)月年齡組(0.95%vs 2.9%,P=0.005,OR=3.047,95%CI:1.396-6.651)。上呼吸感染患者中SAFV的陽(yáng)性率為1.3%,肺炎患者中SAFV的陽(yáng)性率為1.1%,且和非肺炎患者相比,差異具有統(tǒng)計(jì)學(xué)意義(P=0.001,OR=0.308,95%CI:0.152-0.627)。(4)手足口病患者中,具有神經(jīng)系統(tǒng)癥狀的患者SAFV的陽(yáng)性率高于無神經(jīng)系統(tǒng)癥狀的患者(P=0.040,OR=1.475,95%CI:1.016-2.140),重度手足口病患者的陽(yáng)性率高于輕度手足口患者(P=0.021,OR=1.535,95%CI:1.063-2.219)。(5)EV71和SAFV復(fù)合感染與EV71單獨(dú)感染的患者相比較,臨床重癥的發(fā)生率差異具有統(tǒng)計(jì)學(xué)意義,EV71和SAFV復(fù)合感染更容易加重病情(P=0.007)。(6)本研究中通過對(duì)SAFV陽(yáng)性標(biāo)本進(jìn)行VP1片段擴(kuò)增,獲得151株VP1基因序列,系統(tǒng)進(jìn)化分析顯示4種型別的存在:SAFV-1型(17株),SAFV-2型(70株),SAFV-3型(59株),SAFV-6型(5株)。對(duì)各種型別分別進(jìn)行系統(tǒng)進(jìn)化分析,顯示序列具有一定的聚集性和地理分布特征。結(jié)論1.WU多瘤病毒在兒童患者中的流行規(guī)律與基因特征研究(1)首次在手足口病患者中檢測(cè)到WUPy V,其陽(yáng)性率為1.9%。2013年5-7月三種癥候群患者中WUPy V陽(yáng)性率均較高,提示在這一時(shí)間段WUPy V可能出現(xiàn)暴發(fā)。(2)獲得了中國(guó)地區(qū)57株WUPy V全基因組序列。通過系統(tǒng)發(fā)育分析表明,本研究中的序列形成了一個(gè)新的亞支IIIc,序列分布具有地域特征,同時(shí)可能存在地域傳播。手足口病患者糞便標(biāo)本中的10株全基因組序列,其主要型別是Ia型和IIIc型。急性腹瀉患者糞便標(biāo)本中的12株全基因組序列全部為Ia型。(3)WUPy V基因突變不僅存在純化選擇作用,也有正向選擇作用的影響。2.Saffold心病毒在兒童患者中的流行規(guī)律與基因特征研究(1)首次在手足口患者中檢測(cè)到SAFV,其陽(yáng)性率為3.5%,高于呼吸道感染患者和急性腹瀉患者。(2)在手足口病患者中,SAFV可能和神經(jīng)系統(tǒng)癥狀、重度手足口病相關(guān),同時(shí)EV71和SAFV復(fù)合感染可能會(huì)加重手足口病患者的病情。(3)系統(tǒng)進(jìn)化分析顯示,本研究中的SAFV序列分別為SAFV-1型,SAFV-2型,SAFV-3型,SAFV-6型,不同的基因型均具有一定的聚集性和地理分布特征。
[Abstract]:Background Emerging infectious diseases have always threatened human health. The discovery and identification of new viruses and the determination of the relationship between new viruses and diseases are the primary tasks in the prevention, diagnosis and treatment of new viral infectious diseases. Ebola virus, Boca virus and hemipneumonia virus. Although the etiology of disease virus infection has been studied in depth in recent years, there are still some unknown causes of disease, some unknown causes of disease may be caused by undetected viruses. WU polyoma virus is May 2007, the United States University of Washington Medical School Department. Saffold's heart virus was first isolated in 1981 from the stool of an eight-month-old fever patient of unknown origin. In 2007, it was identified as a cardiovirus by non-sequence-dependent single-primer amplification. It is reported that WU polyomavirus and Saaffold heart virus can be detected in nasopharyngeal aspirates, feces and serum of patients, so they may be one of the pathogens causing the disease. As WU polyomavirus and Saaffold heart virus are newly discovered viruses, they have attracted more and more attention in recent years. The epidemic regularity, pathogenicity and genetic characteristics of the two viruses in China are poorly understood. Most of the studies are limited to one disease. The epidemic regularity, clinical characteristics and genetic characteristics of the two viruses are not analyzed. Nasopharyngeal aspirate specimens from patients with respiratory tract infection, fecal specimens from patients with acute diarrhea and hand-foot-mouth disease were tested for pathogens, and the epidemic regularity and gene evolution characteristics of newly discovered WU polyoma virus and Saffold heart virus in children were systematically analyzed, so as to provide further treatment for viral infection and control of the epidemic situation. Methods Nasopharyngeal aspirates, feces of patients with acute diarrhea and feces of patients with hand-foot-mouth disease were collected from the children's Hospital Affiliated to Chongqing Medical University from January 2012 to December 2015. PCR, RT-PCR, Real-Time PCR, Real-Ti were used. The routine viruses detected in nasopharyngeal aspirates of patients with respiratory tract infection include human adenovirus (HAd V), influenza virus (Flu), human rhinovirus (HRV / HEV), respiratory syncytial virus (RSV), hemipneumonia virus (met). APneumovirus (MPV), parainfluenza virus (PIV), human bocavirus (HBo V) and human coronavirus (HCo V). Conventional viruses detected in stool samples from patients with acute diarrhea include rotavirus (Rt V), norovirus (No), adenovirus (Ad V), sapoviruses (Sapoviruses). Enterovirus (EV), Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the common viruses detected in stool specimens from patients with HFMD. Nasopharyngeal aspirate specimens from patients with respiratory tract infections, stool specimens from patients with acute diarrhea, and hand, foot and mouth specimens from patients with HFMD WU polyomavirus (WUPy V) and Saffold Cardiovirus (SAFV) were detected in fecal specimens of the patients. WU polyomavirus positive specimens were amplified by whole genome amplification, Saffold cardiovirus positive specimens were amplified by VP1 fragment amplification, typing and phylogenetic analysis. Epidata 3.1 data were recorded and SPS was used S 20.0 software for statistical analysis, test level alpha = 0.05. Construction of phylogenetic tree using software Mega 7.0, using maximum likelihood hood method, bootstrap value set to 1000 for construction and testing. Selective pressure analysis using FEL, IFEL, MEME, SLAC four methods screening, at least three methods of the same site detection is determined to be a positive choice. Results 1. Epidemiological and genetic characteristics of WU polyoma virus in children (1) This study detected 170 cases of WUPy V positive, the total detection rate was 4.7%. Of them, 127 cases (7.8%) were WUPy V positive in nasopharyngeal aspirates of respiratory tract infection patients. There were 25 (2.4%) WUPy V positive stool specimens from patients with acute diarrhea and 18 (1.9%) WUPy V positive stool specimens from patients with hand-foot-mouth disease. The positive rates of WUPy V were statistically significant (P 0.001). The positive rate of WUPy V in patients with respiratory tract infection was the highest. (2) The combined infection rate of WUPy V was 79.4% (135/170) among the three types of cases. There were 97 cases with one virus infection, 31 cases with two viruses infection, 7 cases with three or more viruses infection and 35 cases with WUPy V infection alone. The most common viruses were Rt V (11 cases), hand-foot-mouth disease (83.4% (15/18) and EV71 (9 cases). (3) According to the time distribution of the positive rate, the positive rate of WUPy V was the highest in patients with respiratory tract infection from May to June 2013, and acute diarrhea and hand-foot-mouth disease (HFMD) had a test in July 2013. The positive rate of WUPy V infection was higher than that of WUPy V infection alone (P 0.05). The infection rate of WUPy V was 11.4% in patients with upper respiratory tract infection. The WUPy V infection rate was 7.8%. (5) 57 WUPy V genome sequences were successfully amplified with homology of 98.7%-100%. Phylogenetic analysis showed that the WUPy V infection rate was significantly different from that of non-upper respiratory tract infection patients (P = 0.018, OR = 1.770, 95% CI: 1.103-2.842). In this study, the sequences were clustered in Ia, Ic and IIC, and a new subbranch IIIc was formed. 35 strains from patients with respiratory tract infection were clustered in Ia (20 strains), Ic (8 strains), and IIC (7 strains). 12 strains from patients with acute diarrhea were all distributed in Ia. Sequences of 10 strains, 8 strains in Ia, 2 strains in IIIc. (6) WUPy V selection pressure analysis showed that VP1 fragment No. 82 was a positive selection site, VP2, VP3, STAg, LTAg were not found positive selection sites. Among them, 44 cases (1.3%) were detected in nasopharyngeal aspirates of patients with respiratory tract infection, 28 cases (0.9%) in feces of patients with acute diarrhea and 118 cases (3.5%) in feces of patients with hand-foot-mouth disease. The positive rates of the three cases were different, and the difference was statistically significant (P 0.001). The combined infection rate was 73.2% (139/190), 116 cases complicated with one virus infection, 18 cases complicated with two viruses infection, 5 cases complicated with three viruses and more, 51 cases infected with SAFV alone. (3) In patients with respiratory tract infection, the positive rate of SAFV in patients older than 36 months was higher than that in 1-6 months (0.95% vs 2.9%, P = 0.005, OR = 3.047, 95% CI: 1.396-6.651). The positive rate of SAFV was 1.3% in patients with upper respiratory infection and 1.1% in patients with pneumonia, and the difference was statistically significant (P = 0.001, OR = 0.308, 95% CI: 0.152-0.627). (4) In patients with HFMD, the positive rate of SAFV in patients with neurological symptoms was higher than that in patients without neurological symptoms (P = 0.040, O = 0, O = 0.040). R = 1.475, 95% CI: 1.016-2.140, and the positive rate of severe HFMD patients was higher than that of mild HFMD patients (P = 0.021, OR = 1.535, 95% CI: 1.063-2.219). (5) Comparing with EV71 and SAFV infection alone, the incidence of severe clinical infections was significantly different. EV71 and SAFV infection were more likely to aggravate the disease (P = 0.007). In this study, 151 VP1 gene sequences were obtained from SAFV-positive specimens. Phylogenetic analysis showed that there were four types: SAFV-1 (17 strains), SAFV-2 (70 strains), SAFV-3 (59 strains) and SAFV-6 (5 strains). Phylogenetic analysis showed that the sequences were clustered and geographically distributed. The positive rate of WUPy V was 1.9%. The positive rate of WUPy V was higher among the three groups of patients from May to July 2013, suggesting that there might be an outbreak of WUPy V in this period. (2) 57 strains of WUPy V were obtained in China. Genomic sequence. Phylogenetic analysis showed that the sequence in this study formed a new subbranch IIIc with regional characteristics and possible regional transmission. 10 complete genomic sequences of fecal samples from patients with hand-foot-mouth disease were of type Ia and type IIIc. 12 of the fecal samples from patients with acute diarrhea were of type Ia and type IIIc. The whole genome sequence was typeIa. (3) WUPy V gene mutation not only had purification selection effect, but also had positive selection effect. (2) Saffold heart virus epidemiology and genetic characteristics in children (1) SAFV was detected in hand, foot and mouth patients for the first time, the positive rate was 3.5%, higher than that in respiratory tract infection patients and acute diarrhea patients. (2) In HFMD patients, SAFV may be associated with neurological symptoms, severe HFMD, and the combined infection of EV71 and SAFV may aggravate the condition of HFMD patients. (3) Phylogenetic analysis showed that the SAFV sequences in this study were SAFV-1, SAFV-2, SAFV-3, SAFV-6, and different genotypes all had certain genotypes. The characteristics of clustering and geographical distribution.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.1;R181.3

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