兰州地区5岁以下婴幼儿病毒性腹泻的分子流行病学研究

目的调查兰州地区5岁以下婴幼儿病毒性腹泻的流行情况,了解四种主要腹泻病毒在儿童中的分布情况。方法采集2009年7月至2010年6月兰州大学第一医院儿科5岁以下腹泻患儿粪便标本290份及儿童保健中心健康婴幼儿正常粪便标本114份,采用酶联免疫吸附试验（ELISA）检测轮状病毒抗原,采用巢式聚合酶链反应对轮状病毒阳性标本进行分型;采用反转录．聚合酶链反应（RT—PCR）检测杯状病毒和星状病毒,聚合酶链反应（PCR）检测腺病毒。结果290份腹泻标本中四种病毒的阳性率分别为：轮状病毒39．31％,杯状病毒11．38％,腺病毒10．69％,星状病毒4．83％;对114份轮状病毒阳性标本G、P分型,G3型及P[8]型为优势株;114份正常标本轮状病毒检出率为0,杯状病毒检出7例,星状病毒检出1例,腺病毒检出5例。结论病毒性病原在兰州地区婴幼儿腹泻中占有重要地位,长期系统的监测具有重要意义。     

深圳市儿童秋冬季腹泻诺如病毒检测及基因型分析

目的 了解深圳市儿童秋冬季腹泻中诺如病毒（Nov）的感染状况,并对阳性株进行基因型分析.方法 收集深圳地区多家医院2009年9月至2010年1月腹泻患儿粪便标本307份,通过逆转录-聚合酶链反应（RT-PCR）方法进行诺如病毒核酸扩增,部分阳性标本的PCR产物经纯化、测序,结合GenBank参考株相应核酸序列构建系统进化树并进行基因型分析,采用SimPlot3.5.1对重组株进行分析和鉴定.结果 307份粪便标本中诺如病毒核酸阳性38例,阳性率为12.4％,以6～24个月龄婴幼儿感染为主,占全部病例数的81.6％（31 /38）,感染的发病高峰为10、11月份,占全部病例数的73.7％ （28/38）;26份测序标本中25株属于GⅡ.4型2006b变异体,1株为GⅡ.12/GⅡ.3型重组株.结论 诺如病毒是深圳市儿童秋冬季腹泻的重要病原体,优势流行株为GⅡ.4型2006b变异体,深圳首次检出的诺如病毒重组株为GⅡ.12/GⅡ.3型.     

太原市5岁以下腹泻住院儿童的病毒病原及其流行特征

目的 了解山西省太原市5岁以下腹泻住院儿童四种主要腹泻病毒的流行情况.方法 收集山西儿童医院2007年10月至2008年11月5岁以下全部住院腹泻患儿的粪便标本,采用ELISA试剂盒来检测轮状病毒;采用聚合酶链反应（PCR）检测腺病毒;逆转录-聚合酶链反应（RT-PCR）法检测星状病毒和杯状病毒并对轮状病毒进行分型.结果 346份标本中轮状病毒占40.8%、杯状病毒占7.5%、星状病毒占6.4%、腺病毒占3.2%.对141份轮状病毒阳性标本进行G1P分型,G1型是最优势株,P型优势株为P[8].四种病毒主要是感染2岁以下婴幼儿,RV有明显的季节的特征,9-11月份（48.92%）为发病高峰.结论 轮状病毒为最主要的病毒病原,G1P[8]型为主要流行株,秋冬季为发病高峰,2岁以下为发病高危人群,混合感染多见.     

南京地区婴幼儿杯状病毒感染的分子流行病学研究

目的 了解南京地区婴幼儿杯状病毒腹泻的感染状况、临床表现以及分子流行病学特征.方法 采集2010年7月至2011年6月南京医科大学附属南京儿童医院5岁以下腹泻患儿粪便标本及儿童保健中心健康婴幼儿粪便标本各428份.采用反转录-聚合酶链反应( RT-PCR)检测杯状病毒,测序确定其基因型别.结果 428份腹泻样本中有63份为杯状病毒阳性,检出率为14.72％.其中诺如病毒GⅡ型58例,未检出诺如病毒GⅠ型,札如病毒5例,以诺如病毒GⅡ-4 2006b型为主要流行株.428份健康对照组标本杯状病毒检出19例,诺如病毒6例,札如病毒11例,2例为诺如病毒GⅡ型和札如病毒混合感染.结论 南京地区婴幼儿中存在不同基因型杯状病毒感染,流行毒株以GⅡ.2006b为主.     

江苏省苏州及南京地区2010年婴幼儿腹泻中诺如病毒分子流行病学研究

目的 了解江苏地区婴幼儿腹泻患者中诺如病毒的感染情况,明确该地区诺如病毒的主要基因型别和流行病学特征.方法 收集2010年江苏省苏州市婴幼儿医院及南京市婴幼儿医院疑似病毒性腹泻粪便标本498例,采用荧光定量PCR方法检测粪便中诺如病毒RNA及其基因组别,并对部分阳性标本测序以明确基因型.结果 498份粪便标本中,GⅠ组诺如病毒阳性标本为2例,阳性率为0.4％,GⅡ组诺如病毒为190例,阳性率为38％,对本地区部分诺如病毒阳性标本进行测序,2份GⅠ标本中,一株为GⅠ1型,另一株为GⅠ3型;测序的23份GⅡ标本中21株为GⅡ4型,2株为GⅡ3型.结论 诺如病毒是本省婴幼儿病毒性腹泻的重要病原体,以诺如病毒GⅡ4型为主,同时也存在其他型别的感染流行.     

2011年苏州地区诺如病毒GⅡ组变异株的分子特征研究

目的 了解苏州地区诺如病毒的感染状况及其变异株的分子特征.方法 收集苏州市儿童医院疑似病毒性腹泻粪便标本419例,采用荧光定量PCR方法检测粪便中诺如病毒RNA及其基因组别,并对部分阳性标本测序分析.结果 419份粪便标本中,GⅡ组诺如病毒为122例,阳性率为29％,未检测出GⅠ组诺如病毒,对本地区部分诺如病毒阳性标本进行测序,A区(RdRp区)测序的25份GⅡ标本中25株均为GⅡ.4型;C区(N-S区)测序的26份GⅡ标本中17株为GⅡ.4型,8株为GⅡ.3型,1株为GⅡ.2型;D区(P区)测序的25份GⅡ标本中16株为GⅡ.4型,9株为GⅡ.3型.结论 诺如病毒是本地区婴幼儿病毒性腹泻的重要病原体,以诺如病毒GⅡ.4-2006b亚型为主,此外发现有GⅡ.4-2010亚型(GⅡ.4 New Orleans株)的流行,这是国内首次对该亚型毒株进行报道,同时还检测到部分重组毒株,提示诺如病毒在本地区的遗传变异日趋复杂.     

南京地区婴幼儿杯状病毒感染的分子流行病学研究

目的了解南京地区婴幼儿杯状病毒腹泻的感染状况、临床表现以及分子流行病学特征。方法采集2010年7月至2011年6月南京医科大学附属南京儿童医院5岁以下腹泻患儿粪便标本及儿童保健中心健康婴幼儿粪便标本各428份。采用反转录-聚合酶链反应（RT—PCR）检测杯状病毒,测序确定其基因型别。结果428份腹泻样本中有63份为杯状病毒阳性,检出率为14．72％。其中诺如病毒GⅡ型58例,未检出诺如病毒GI型,札如病毒5例,以诺如病毒GⅡ-42006b型为主要流行株。428份健康对照组标本杯状病毒检出19例,诺如病毒6例,札如病毒11例,2例为诺如病毒GⅡ型和札如病毒混合感染。结论南京地区婴幼儿中存在不同基因型杯状病毒感染,流行毒株以GⅡ-2006b为主。     

人偏肺病毒感染住院儿童的临床特征及病毒基因特性分析

目的 了解急性呼吸道感染住院儿童中人偏肺病毒(hMPV)的感染情况、临床特征及人偏肺病毒基因特性.方法 对2006年2月至5月、2008年3月至4月及2008年9月至2009年2月收集的天津市儿童医院急性呼吸道感染住院儿童的310份鼻咽吸取物标本进行巢式PCR检测hMPV N基因片段,并对其中17株N基因片段进行序列测定,分析序列特征并绘制种系发生树.收集所有阳性患者的临床资料.同时采用多重PCR对N基因阳性标本进行其他常见呼吸道病毒的检测.结果 310份标本中共检出20份(6.5%)hMPV.hMPV阳性患儿的年龄中位数为15.0个月(16天～9岁),其中≤2岁患儿占90.0%(18/20).男性占60.0%(12/20).17株N基因种系发生树分析显示,11株为A2b亚型.20例hMPV阳性患儿的临床诊断均为支气管肺炎,占所有采集的肺炎患儿标本的7.1%(20/282),其临床症状以咳嗽、喘息、呼吸急促、发烧等常见.35%(7/20)需要重症监护,15%(3/20)需要吸氧治疗.平均住院时间为(11.9±4.8)d.A、B型间临床特征未见明显差别.hMPV无明显流行高峰.2例hMPV阳性患儿分别与腺病毒和鼻病毒混合感染.结论 hMPV可引起婴幼儿肺炎等严重疾病.天津地区流行的hMPV以A2b为优势流行型.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的 调查烟台市沿海地区人源与猪源戊型肝炎病毒(HEV)基因型别的相关性.方法 应用逆转录一巢式聚合酶联反应( RT-nPCR)方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEV RNA检测,并对HEV RNA阳性标本进行克隆测序和序列分析.结果 16例急性散发戊型肝炎患者中有7例粪便标本HEV RNA阳性;51份lgM阳性正常人群血清标本中有1份HEV RNA阳性;34份猪胆汁标本中有1份HEV RNA阳性.序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为87％～ 98.1％.7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％ ～ 98.1％之间;其中有6例患者和猪的基因序列同源性在93.9％ ～98.1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型.正常人群的1例戊肝病毒基因型为Ⅰ型d亚型.该地区人与猪HEV的ORF2的部分基因片段与HEV Ⅰ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82.5％～100％,81.7％ ～92.9％,81.4％ ～93.9％,84.9％ ～ 100％.结论 该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEV Ⅰ型在人群中散在存在.     

腹泻住院患儿中检测到人双埃可病毒

目的 在腹泻住院患儿粪便标本中检测人双埃可病毒(HPeV),探讨HPeV与婴幼儿重症腹泻之间的联系.方法 通过Real-time PCR直接从<5岁未知病原住院腹泻患儿粪便标本中检测HPeV,进而通过基于VP3/VP1连接区的巢式PCR扩增片段的测序结果进行分型.结果 99份腹泻粪便标本,HPeV阳性24例(24%),其中,以HPeV1为主(50%),其次为HPeV3(25%),HPeV4(8.3%),HPeV6(4.2%),还有3例HPeV阳性标本未能分型,未检测到HPeV2、5、7、8.HPeV感染的住院腹泻患儿主要为<1岁患儿.HPeV感染高峰为秋冬季节,10月达到峰值.VP3/VP1连接区基因与已知HPeV的病因构成及深入了解HPeV的病原学及流行病学特点有重要意义.     

Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification

In vitro enzymatic amplification was applied to detect hepatitis B virus (HBV) DNA sequences in serum. This technique, known as the polymerase chain reaction (PCR) was used to amplify a 128 bp DNA fragment including a 112 nucleotide long sequence complementary to a region in the S gene of the HBV genome. Amplified samples were subjected to spot-test hybridization and scintillation counting using a 32P-labeled oligonucleotide probe. A kinetic study, performed for 4 to 32 PCR cycles with a viral particle preparation, showed a time-limited exponential accumulation of the specific amplified DNA fragment. Amplification yield after 32 cycles was at least 4 X 10(6) with a detection limit equal to 3 X 10(2) viral particles per ml of serum. As the reliability of the PCR technique was greatest for 24 PCR cycles, these conditions were used to develop a quantitative test with a detection limit of 4 X 10(4) viral particles per ml of serum. Results of this test were perfectly correlated with those obtained from the classical spot test without amplification. Ethidium bromide stained agarose gel and Southern blot analysis confirmed the specific amplification of the 128 bp HBV DNA fragment.     

Molecular characterization of the first Aichi viruses isolated in Europe and in South America

Summary. The occurrence of Aichi virus, a picornavirus associated with acute gastroenteritis, has so far only been described in Asian countries. This study reports the first finding of Aichi virus in clinical specimens from Germany and Brazil. The nucleotide sequences of both a German and a Brazilian isolate were determined, analyzed, and compared to known Aichi sequences. The German strain turned out to be a member of genogroup A, while the Brazilian belonged to genogroup B. For a primary assessment of the epidemiological importance of Aichi virus in Germany, a panel of 485 German serum samples was screened for antibody to Aichi virus, and a seroprevalence of 76% was found. First two authors contributed equally.     

江门成人散发急性腹泻者星状病毒的检测及毒株型别分析

目的研究江门地区成人腹泻者星状病毒感染情况及型别特点。方法收集江门地区病毒性腹泻流行期散发成人腹泻患者粪便标本，经胶体金法、RT-PCR法分别检测轮状病毒和诺如病毒后，运用实时荧光PCR进行星状病毒检测．并对阳性标本进行RT—nested PCR测序，鉴定型别。结果56份成人病毒性腹泻粪便标本中．荧光PCR法测出3份星状病毒株（5．4％），经RT—PCR测序1份，序列分析鉴定为HAtsV-4型。结论江门成人散发病毒性腹泻者存在星状病毒感染，型别为HAstV-4，未发现多致泻病毒混合感染。     

Strategy for identifying the gene encoding the DNA polymerase of molluscum contagiosum virus type 1

Molluscum contagiosum virus (MCV) is a member of the family Poxviridae and pathogenic to humans. MCV causes benign epidermal tumors mainly in children and young adults and is a common pathogen in immunecompromised individuals. The viral DNA polymerase is the essential enzyme involved in the replication of the genome of DNA viruses. The identification and characterization of the gene encoding the DNA polymerase of molluscum contagiosum virus type 1 (MCV-1) was carried out by PCR technology and nucleotide sequence analysis. Computer-aided analysis of known amino acid sequences of DNA polymerases from two members of the poxvirus family revealed a high amino acid sequence homology of about 49.7% as detected between the DNA polymerases of vaccinia virus (genus Orthopoxvirus) and fowlpoxvirus (genus Avipoxvirus). Specific oligonucleotide primers were designed and synthesized according to the distinct conserved regions of amino acid sequences of the DNA polymerases in which the codon usage of the MCV-1 genome was considered. Using this technology a 228 bp DNA fragment was amplified and used as hybridization probe for identifying the corresponding gene of the MCV-1 genome. It was found that the PCR product was able to hybridize to theBamHI MCV-1 DNA fragment G (9.2 kbp, 0.284 to 0.332 map units). The nucleotide sequence of this particular region of the MCV-1 genome (7267 bp) between map coordinates 0.284 and 0.315 was determined. The analysis of the DNA sequences revealed the presence of 22 open reading frames (ORFs-1 to-22). ORF-13 (3012 bp; nucleotide positions 6624 to 3612) codes for a putative protein of a predicted size of 115 kDa (1004 aa) which shows 40.1% identity and 35% similarity to the amino acid sequences of the DNA polymerases of vaccinia, variola, and fowlpoxvirus. In addition significant homologies (30% to 55%) were found between the amino acid sequences of the ORFs 3,-5,-9, and-14 and the amino acid sequences of the E6R, E8R, E10R, and a 7.3 kDa protein of vaccinia and variola virus, respectively. Comparative analysis of the genomic positions of the loci of the detected viral genes including the DNA polymerases of MCV-1, vaccinia, and variola virus revealed a similar gene organization and arrangement.     

海南岛两株甲病毒基因组3′末端核苷酸序列的克隆与分析

目的　从分子水平鉴定海南省分离的两株甲病毒 (HBb17和M1病毒 )。方法　采用RT-PCR方法 ,分别扩增两株病毒基因组的 3′末端核苷酸序列 ,扩增产物经亚克隆 ,筛选重组子并测定核苷酸序列对序列进行分析。结果　HBb17和M1病毒分别扩增出约 1 6kb和 1 3kb的特异片段。序列分析表明 ,在 3′末端非翻译区HBb17病毒与罗斯河病毒 (国际标准株T48病毒 )核苷酸同源性为99 % ,M1病毒与鹭山病毒 (SAG)同源性为 98% ;两病毒结构基因的E1区序列 ,HBb17病毒与罗斯河病毒核苷酸 (氨基酸 )同源性为 99% (99% ) ,M1病毒与鹭山病毒核苷酸 (氨基酸 )同源性为 97% (99% ) ,M1病毒与盖塔病毒同源性为 94% (98% )。结论　序列分析表明 ,海南岛分离的HBb17病毒属于罗斯河病毒 ,M1病毒可能是鹭山病毒或盖塔病毒的变异株     

海南岛两株甲病毒基因组3＇末端核苷酸序列的克隆与分析

目的 从分子水平鉴定海南省分离的两株甲病毒（ＨＢｂ１７和Ｍ１病毒）。方法 采用ＲＴ－ＰＣＲ方法,分别扩增两株病毒基因组的３’末端核苷酸序列,扩增产物经亚克隆,筛选重组子并测定核苷酸序列对序列进行分析。结果 ＨＢｂ１７和Ｍ１病毒分别扩增出约１．６ｋｂ和１．３ｋｂ的特异片段。序列分析表明,在３’末端非翻译区ＨＢｂ１７病毒和Ｍ１病毒与罗斯病毒（国际标准株Ｔ４８病毒）核苷酸同源性为９９％,Ｍ１病毒与鹭山病     

Identification and nucleotide sequence analysis of the repetitive DNA element in the genome of fish lymphocystis disease virus

The genome of the fish lymphocystis disease virus (FLDV) was screened for the existence of repetitive DNA sequences using a defined and complete gene library of the viral genome (98 kbp) by DNA-DNA hybridization, heteroduplex analysis, and restriction fine mapping. A repetitive DNA sequence was detected at the coordinates 0.034 to 0.057 and 0.718 to 0.736 map units (m.u.) of the FLDV genome. The first region (0.034 to 0.057 m.u.) corresponds to the 5' terminus of the EcoRI FLDV DNA fragment B (0.034 to 0.165 m.u.) and the second region (0.718 to 0.736 m.u.) is identical to the EcoRI DNA fragment M of the viral genome. The DNA nucleotide sequence of the EcoRI FLDV DNA fragment M was determined. This analysis revealed the presence of many short direct and inverted repetitions, e.g., a 18-mer direct repetition (TTTAAAATTTAATTAA) that started at nucleotide positions 812 and 942 and a 14-mer inverted repeat (TTAAATTTAAATTT) at nucleotide positions 820 and 959. Only short open reading frames were detected within this region. The DNA repetitions are discussed as sequences that play a possible regulatory role for virus replication. Furthermore, hybridization experiments revealed that the repetitive DNA sequences are conserved in the genome of different strains of fish lymphocystis disease virus isolated from two species of Pleuronectidae (flounder and dab).     

Development of degenerate and species-specific primers for the differential and simultaneous RT-PCR detection of grapevine-infecting nepoviruses of subgroups A, B and C

Based on the nucleotide sequence homology of RNA-1 and RNA-2 of nepoviruses isolated from grapevines, three sets of degenerate primers, one for each of the three subgroups of the genus (A, B and C), were designed and proved effective for RT-PCR detection of subgroups in infected grapevines and herbaceous hosts. Primers designed specifically for detecting subgroup A species amplified a fragment of 255 bp from samples infected by Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV) and Grapevine deformation virus (GDefV), but not from samples infected by other nepovirus species. Similarly, primers for detection of subgroup B nepoviruses amplified a 390 bp product from samples infected by Grapevine chrome mosaic virus (GCMV), Tomato black ring virus (TBRV), Grapevine Anatolian ringspot virus (GARSV) and Artichoke Italian latent virus (AILV). The third set of primers amplified a 640 bp fragment, only from samples infected by subgroup C nepoviruses, i.e Tomato ringspot virus (ToRSV) Grapevine Bulgarian latent virus (GBLV), and Grapevine Tunisian ringspot virus (GTRSV). These primers were able to detect simultaneously all viral species belonging to the same subgroup and to discriminate species of different subgroups. Multiplex-PCR detection of subgroup A and B nepoviruses was obtained using a specific primer (sense for subgroup A and antisense for subgroup B) for each of the species of the same subgroup in combination with the degenerate subgroup-specific primers. In this way it was possible to detect four different viral species in single samples containing mixtures of viruses of the same subgroup. In particular, for viruses of subgroup A (TRSV, GFLV, ArMV and GDefV) amplicons of 190, 259, 301 and 371 bp were obtained, whereas amplicons of 190, 278, 425 and 485 bp, respectively, were obtained from samples infected with viruses of subgroup B (GCMV, AILV, GARSV and TBRV).