博卡病毒属基因组特征与致病的分子机制

博卡病毒是细小病毒科细小病毒亚科的成员之一.目前已知博卡病毒成员有牛博卡病毒、犬博卡病毒、人博卡病毒、以及新鉴定的猪博卡病毒,猩猩、猫、犬以及加利福尼亚海狮体内发现的新博卡病毒成员.作为新发病原,博卡病毒成为各国科研人员的研究热点.本文结合前人的文献和我们近期的研究成果,对博卡病毒的家族成员分类、基因组结构与复制、临床致病特点、致病分子机制等方面进行了阐述,为广大科研人员对博卡病毒的研究提供一定的帮助.     

人博卡病毒及其在中国的流行现状

人博卡病毒(Human bocavirus,HBoV)属于细小病毒科,博卡病毒属。HBoV是除细小病毒B19和人细小病毒4(Human parvovirus,PARV4)外,目前所发现的与人类疾病有关的细小病毒之一。至今已有4种不同的HBoV相继报道,分别为HBoV1、HBoV2、HBoV3和HBoV4。HBoVs感染的发生率差异较大,且患者的临床表现各不相同,常与其它病原体共检出。本文就有关HBoVs的报道,从HBoVs的生物学性状、流行特征、致病机制、系统进化分析及其在我国的流行现状进行了阐述和讨论。     

人博卡病毒I型的研究进展

人博卡病毒1型(HBoV1)为引发呼吸道感染一种新发病毒,具有典型的细小病毒科病毒基因组特征,3个开放阅读框分别编码非结构蛋白NS1、NP1和结构蛋白VP1和VP2;HBoV1进行滚环复制时存在复制环形附加体,附加体的发现为扩增HBoV1全基因组和构建感染性克隆拯救病毒提供可能,同时HBoV1与HBoV2-4间存在着重组关系;HBoV1的体外增殖随着三维立体细胞培养而成为现实,为HBoV1的致病机制研究提供有力平台。本文重点对HBoV1的分子生物学特征、疾病相关性、体外增殖培养、HBoV1的诊断和治疗进行阐述。     

猪博卡病毒的研究进展

猪博卡病毒是细小病毒科细小病毒亚科博卡病毒属的新成员,2009年发现于患仔猪断奶后多系统衰竭综合征的瑞典猪群中。目前,猪博卡病毒作为新发现的病原,已是世界各国科研人员的研究热点。本文结合已发表的文献,对猪博卡病毒的发现、分类、基因组结构与复制、流行病学、与疾病的相关性、培养与检测等方面进行了阐述,为广大科研人员对猪博卡病毒的研究提供一定的理论依据。     

人博卡病毒编码蛋白功能研究

人博卡病毒(Human bocavirus,HBoV)是一类新发现的病毒,属于细小病毒科。至今己发现有4个基因型别(HBoV 1～4),不同基因型别的HBoV临床意义不同,有单一HBoV1感染致死的报道。但迄今为止,其复制机制、致病机理等尚不明确。HBoV基因组全长约为5.5kb,主要表达非结构蛋白NS1、NS2、NS3、NS4和NP1,以及结构蛋白VP1、VP2和VP3。本文旨在对近年来的HBoV各编码蛋白功能研究进展进行综述,并对HBoV未来可能的研究方向进行展望。     

一株水貂博卡病毒全基因组扩增及序列分析

本研究旨在研究水貂博卡病毒（Mink bocavirus,MBoV）在山东省的流行状况及基因特征。采用PCR对采自山东省境内水貂养殖场的85份水貂腹泻样品进行MBoV检测,挑选1份单阳性样品进行全基因扩增,使用MegAlign进行序列同源性比对分析,利用DNAMAN V6对基因组5’末端和3’末端回文结构进行预测,应用MEGA V6进行遗传进化分析。结果表明,所采集的腹泻样品中MBoV阳性率为3.5%(3/85),其中1份为MBoV单阳性,2份为MBoV与水貂肠炎病毒（Mink enteritis virus,MEV）双阳性。获得MBoV全基因序列1条（SDMBoV2020）,全长5 252 bp;经分析,5’-和3’-UTR分别由98和145 bp短回文序列组成,具有典型的细小病毒末端的茎环样结构;基因组含有3个ORFs,分别编码非结构蛋白NS1、NP1以及结构蛋白VP1和VP2;SDMBoV2020与NCBI登录的MBoV参考毒株KU950356各编码基因的推导氨基酸序列同源性较高,为98.5%～99.0%;基于MBoV全基因序列构建的系统发育进化树也显示,SDMBoV2020和参...     

WLL-1株博卡病毒(Bocavirus)全基因组序列分析

儿童下呼吸道感染已成为当前儿科发病率最高的一种疾病,而病毒是小儿下呼吸道感染的重要原因。临床上有相当比例的小儿下呼吸道感染病因未能作出明确的实验室诊断,给临床诊断与治疗带来了较大的困难。小儿下呼吸道感染可以由不同病毒引起,其中包括呼吸道合胞病毒、流感病毒、腺     

博卡病毒的检测方法概述

自2005年博卡病毒发现以来,先后在呼吸道和肠道标本中检出博卡病毒1~4型。博卡病毒1主要与呼吸道感染相关,博卡病毒2~4型主要与肠道感染相关。博卡病毒感染具有典型的咳嗽、喘息、肺炎和腹泻等临床症状,然而由于博卡病毒缺乏合适的宿主细胞而难以培养限制其致病机理等相关研究。立体上皮细胞培养平台、反向遗传学和病毒宏基因组学作为新一代技术有望成为研究博卡病毒致病机理和发现新病毒的有力工具。本文针对博卡病毒已有的电镜、细胞培养、PCR和免疫学检测方法进行综述,以期为博卡病毒的深入研究提供方法参考。     

人博卡病毒基因克隆及衣壳编码基因序列变异分析

为了解人博卡病毒(Human Bocavirus,HBoV)VP1基因进化关系;阐明HBoV目前具体的变化规律,用PCR的方法扩增了1株HBoV的全基因和9株HBoV的VP1基因,克隆并测序,在此基础上,将HBoV的全基因序列和衣壳序列分别与细小病毒亚科其他14个有代表性的病毒进行遗传分析,构建进化树,对目前所有可得到的HBoV的17个衣壳蛋白进行二级结构分析和抗原性分析。结果显示:HBoV全基因序列与B19关系较远,但衣壳序列遗传关系较近。以有典型性的猫瘟细小病毒(Feline parvovirus,FPV)衣壳蛋白为参照,分析多个HBoV衣壳序列之间的变异情况,显示HBoV衣壳的二级结构基础表现出较高的保守性,序列之间的变化主要发生在高抗原区域和感染活性区域。衣壳病毒变异情况显示HBoV在稳定自身的情况下表现出一定的活跃性以逃避免疫反应,也表现出一定的感染适应力。     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

Identification and Characterization of a New Bocavirus Species in Gorillas

A novel parvovirus, provisionally named Gorilla Bocavirus species 1 (GBoV1), was identified in four stool samples from Western gorillas (Gorilla gorilla) with acute enteritis. The complete genomic sequence of the new parvovirus revealed three open reading frames (ORFs) with an organization similar to that of known bocaviruses. Phylogenetic analysis using complete capsid and non structural (NS) gene sequence suggested that the new parvovirus is most closely related to human bocaviruses (HBoV). However, the NS ORF is more similar in length to the NS ORF found in canine minute virus and bovine parvovirus than in HBoV. Comparative genetic analysis using GBoV and HBoV genomes enabled characterization of unique splice donor and acceptor sites that appear to be highly conserved among all four HBoV species, and provided evidence for expression of two different NS proteins in all primate bocaviruses. GBoV is the first non-human primate bocavirus identified and provides new insights into the genetic diversity and evolution of this highly prevalent and recently discovered group of parvoviruses.     

Direct Species Identification of Common Pathogenic Dermatophyte Fungi in Clinical Specimens by Semi-nested PCR and Restriction Fragment Length Polymorphism

OBJECTIVE: To seek a rapid and reliable molecular biology method to identify the common pathogenic dermatophyte fungi from clinical samples. METHOD: The genome DNA was extracted from cultured strains of seven common dermatophyte fungi species and part of each positive clinical specimen by microscopy. Intergenic spacer regions of ribosomal DNA (ITS) were amplified by semi-nested PCR (snPCR) with three universal primers (NS5, ITS1, and ITS4) for fungi. The amplified products were digested with two restriction endonucleases (BciT130 I, Dde I), the Restriction Fragment Length Polymorphism(RFLP). The rest of each clinical specimen was cultured in Sabouraud's Agar medium. Then the results of RFLP were compared with the traditional culture results. RESULTS: The digestion of seven common dermatophyte fungi produced seven different restriction profiles. Restriction profiles of 17 clinical specimens matched, respectively, to that of the cultured strains, and 14 profiles of the 17 ones matched the culture result completely. The coincidence was 100.0%. CONCLUSIONS: snPCR-RFLP analysis of intergenic spacer regions of ribosomal DNA is a valuable method of exactness and clarity for species identification of common dermatophyte fungi from clinical specimens.     

大豆油中转基因成分的Nested PCR和Semi-nested PCR检测方法研究

对大豆油中DNA提取方法进行了研究,结果表明CTAB、SDS和Wizard试剂盒提取大豆油DNA均具有良好的效果。利用nested PCR和semi—nested PCR检测大豆（Roundup Ready）油中的转基因成分发现,该方法能够检测到大豆原油中的Lectin基因（112bp）、CaMV35S基因（147bP）和CP4-EPSPS基因（205bp）,检测灵敏度达到10^-6ng／μl;但该方法未能从人豆成品油（一级）中扩增到上述基因,当中的转基因成分DNA含量低于10^-12ng／μl。     

Porcine Bocavirus: A 10-Year History since Its Discovery

Virologica Sinica - Porcine bocavirus (PBoV) is a single-stranded DNA virus, belongs to the genus Bocaparvovirus of family Parvoviridae. It was discovered along with porcine circovirus 2 (PCV 2)...     

Complete genome sequence of a novel species of Porcine Bocavirus, PBoV5

Porcine bocavirus 5 is a novel porcine bocavirus species found in a pig with clinical diarrhea from a farm in China. Here, we report the complete genome sequence of strain PBoV5/JS677, which will help toward understanding the molecular and evolutionary characteristics of the porcine bocavirus.     

Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

Nuclear reprogramming of somatic cells can be induced by oocyte factors. Despite numerous attempts, the factors responsible for successful nuclear reprogramming remain elusive. In the present study, we found that porcine oocytes with the first polar body collected at 42 h of in vitro maturation had a stronger ability to support early development of cloned embryos than porcine oocytes with the first polar body collected at 33 h of in vitro maturation. To explore the key reprogramming factors responsible for the difference, we compared proteome signatures of the two groups of oocytes. 18 differentially expressed proteins between these two groups of oocytes were discovered by mass spectrometry (MS). Among these proteins, we especially focused on vimentin (VIM). A certain amount of VIM protein was stored in oocytes and accumulated during oocyte maturation, and maternal VIM was specifically incorporated into transferred somatic nuclei during nuclear reprogramming. When maternal VIM function was inhibited by anti-VIM antibody, the rate of cloned embryos developing to blastocysts was significantly lower than that of IgG antibody-injected embryos and non-injected embryos (12.24 versus 22.57 and 21.10%; p < 0.05), but the development of in vitro fertilization and parthenogenetic activation embryos was not affected. Furthermore, we found that DNA double strand breaks dramatically increased and that the p53 pathway was activated in cloned embryos when VIM function was inhibited. This study demonstrates that maternal VIM, as a genomic protector, is crucial for nuclear reprogramming in porcine cloned embryos.     

First Identification and Characterization of Porcine Enterovirus G in the United States

Porcine enterovirus G (EV-G) is a member of the family Picornavirdae, genus Enterovirus. To date, eleven EV-G types (EV-G1 through EV-G11) have been identified in pigs from Asia and Europe however they have never been reported in North America. In this study, we isolated and characterized the complete genome of NP/2013/USA, an EV-G from a porcine diarrhea sample from the United States. The complete genome consists of 7,390 nucleotides excluding the 3′ poly(A) tail, and has an open reading frame that encodes a 2,169 amino acid polyprotein. NP/2013/USA was most similar at the nucleotide (84%) and amino acid (95%) level to the {"type":"entrez-nucleotide","attrs":{"text":"HM131607","term_id":"321228163","term_text":"HM131607"}}HM131607, an EV-G1 type isolated from China in 2012.     

猪肝细胞和培养上清液中猪内源性逆转录病毒的检测

建立了猪肝细胞及其培养上清液中猪内源性逆转录病毒(PERV)的检测方法,探讨了其在猪肝细胞生物人工肝应用中的意义。以PERV gag基因为靶序列,选用特定的引物,PCR检测中国实验用小型猪肝细胞PERV前病毒DNA;RT-PCR检测猪、犬、大鼠以及HBV阳性病人血清和猪肝细胞培养6h、24h时的上清液PERV RNA,同时检测猪肝细胞猪线粒体DNA(mtDNA)。研究结果表明：检测5份中国实验用小型猪血清、肝细胞及培养猪肝细胞24h时的上清液PERV均为阳性,而5份培养猪肝细胞6h时的上清液、5份犬血清、5份大鼠血清和5份HBV阳性病人血清PERV检测结果均为阴性,猪肝细胞中均可检测到猪mtDNA。因此,中国实验用小型猪肝细胞携带PERV;PERV可释放到血清中;猪肝细胞培养24h后该病毒颗粒已释放到培养液中;PCR和RT-PCR方法检测PERV具有特异性强、简便的特点。