中国浙江地区儿童下呼吸道感染KI多瘤病毒的鉴定

呼吸道病毒感染可以导致大面积人群致病[1],近些年,临床上由于不明原因导致的小儿下呼吸道感染病例屡见不鲜,对于该类患儿一般采取抗生素混合治疗的方法,但是治疗效果不佳.尽管过去几年开展的多项研究已经鉴定到了很多的病原体,如腺病毒,鼻病毒属病毒,冠状病毒属病毒,呼吸道合胞体病毒,流感病毒,副流感病毒等.     

单克隆抗体在呼吸道合胞病毒感染诊断和防治中的应用

呼吸道合胞病毒 (RSV)是世界范围内婴幼儿下呼吸道感染的重要病原 ,目前尚无成熟疫苗应用于临床。展开对RSV被动免疫的研究显得尤为重要。单克隆抗体成为深入研究和防治RSV感染的有力武器。本文综述了单克隆抗体在RSV感染的诊断、预防和治疗中的应用     

细菌抗原测定在小儿下呼吸道感染诊断上的应用

<正> 在冬季,急性下呼吸道感染不仅是小儿发生疾病的主要原因,而且是儿科医生诊所最常见的就诊者。各种呼吸道病毒和支原体是下呼吸道感染的重要的病原微生物,它们在小儿患者中的流行病学特征文献已有详尽描述。而要从下呼吸道中把细菌分离出来则是比较困难的,这是由于无症状的化脓菌带菌者特多,故口咽拭子细菌培养与肺组织培     

广州地区小儿呼吸道合胞病毒肺炎病原学研究

本文报告了广州地区小儿病毒性肺炎275例中经病毒分离和／或血清学检查证实病原为呼吸道合胞病毒(RSV)的82例,阳性率为29．8％。首次在广州地区证实了RSV与小儿肺炎的病原关系。 RSV是本地区一岁以内婴儿最重要的下呼吸道感染病原,流行高峰为夏秋季,但病例终年不断,与我国北方流行情况正好相反,具有亚热带地区的流行特点。本文并对实验室诊断方法作了探讨。     

北京地区住院患儿WU多瘤病毒感染的流行病学研究

自2007年Gaynor A M等人通过高通量测序在肺炎患儿呼吸道样本中发现了WU多瘤病毒(Washington University polyomavirus,WUPyV)以来,WUPyV在世界各地被广泛检出。为了解北京地区急性呼吸道感染住院儿童中WUPyV感染情况及其临床特征,收集北京地区2017年4月至2018年3月共1 276份呼吸道感染住院患儿的鼻咽抽吸物样本,使用real time PCR方法对样本进行WUPyV检测,同时对WUPyV阳性样本进行17种常见呼吸道病毒筛查。共检出WUPyV阳性样本76份(5.96%,76/1 276),4岁以下儿童居多(92.11%,70/76);WUPyV感染全年可见,无显著季节性;多伴随其他呼吸道病毒混合感染(60.53%,46/76),最常见混合感染为鼻病毒和A型流感病毒;WUPyV单一感染者与混合感染者病毒载量无显著性差异,临床诊断和表现基本一致;WUPyV感染患儿的常见诊断为支气管炎(68.42%,52/76)和肺炎(30.26%,23/76);临床症状主要表现为高热、咳嗽、咳痰。研究结果提示WUPyV是北京地区急性呼吸道感染住院患儿呼吸道样本中常见病毒之一,多见于4岁以下儿童。     

ELISA检测RSV感染的研究

呼吸道合胞病毒(RSV)是婴幼儿呼吸道感染的重要病原之一,早期快速诊断RSV感染是防止本病蔓延,及时采取正确治疗措施的关键;所以,急待寻求快速诊断的方法。本试验建立了酶联免疫吸附测定(ELISA)检测RSV感染的方法。对其敏感性、特异性、重复性进行了检验;并在临床应用上与病毒分离和/或补体     

人博卡病毒感染的研究进展

细小病毒科属于DNA病毒,其中的细小病毒亚科可进一步分成五个种属,大多数细小病毒属主要感染家畜并致病。直到2005年,研究人员认为B19是唯一的一个对人类致病的细小病毒属病毒。但是2005年10月瑞典科学家Al-lander等从小儿下呼吸道感染分泌物中发现一种新的细小病毒,该病毒能引     

副流感病毒的研究进展

副流感病毒是一类有包膜的单股负链RNA病毒,属副黏病毒,是一种常见的易被忽略的呼吸道感染病原体,主要引起婴幼儿及儿童严重的下呼吸道感染,其致病性仅次于呼吸道合胞病毒(RSV).目前,对副流感病毒的感染,缺乏有效的治疗药物和病毒疫苗.因此,建立快速、敏感和特异的副流感病毒感染的诊断方法,研发新型抗病毒药物和安全有效的副流感病毒疫苗成为研究热点.本研究就副流感病毒的生物学特性、疫苗研究、防治及检测技术作一综述.     

小儿下呼吸道感染的常见病原体及发病机制

引起小儿下呼吸道感染的常见病原体有肺炎链球菌、流感嗜血杆菌、金黄色葡萄球菌,卡他莫拉菌和肺炎支原体等。了解各种病原体的病原学特点及发病机制,并及时、正确的治疗,对临床防治下呼吸道感染具有重要意义。     

呼吸道感染住院患儿Merkel细胞多瘤病毒检测及临床研究

目的:了解北京地区Merkel细胞多瘤病毒(MCPyV)在呼吸道感染住院儿童中的流行情况和临床特点。方法:采集北京友谊医院儿科呼吸道感染住院患儿的鼻咽抽吸物样本200份,并收集相应的患儿临床资料,采用TaqMan real-time PCR方法检测MCPyV LTAg基因,并经测序确认;MCPyV阳性样本同时检测常见的人呼吸道病毒混合感染情况。结果:200份标本中共检出MCPyV阳性6例,检出率3%;感染儿童年龄从6月到5岁,其中年龄≤3岁的占83.3%(5/6)。诊断包括支气管肺炎和急性支气管炎,临床表现包括发热、咳嗽、喘息。6例阳性样本均与其他呼吸道病毒混合感染,A型流感病毒和呼吸道合胞病毒混合感染最常见,6例阳性样本MCPyV载量均低于10拷贝/μL。结论:real-time PCR方法检测呼吸道感染住院患儿中MCPyV的感染率为3%,6例MCPyV阳性样本均与其他呼吸道病毒混合感染且MCPyV载量较低,不能认为MCPyV是儿童呼吸道感染的病因。     

Identification and nearly full-length genome characterization of novel porcine bocaviruses

The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7-56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A(2) (sPLA(2)) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca(2+)-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.     

Ungulate bocaparvovirus 4 and rodent bocavirus are different genotypes of the same species of virus

Bocaviruses are associated with many human infectious diseases, such as respiratory tract infections, gastroenteritis, and hepatitis. Rats are known to be reservoirs of bocaviruses, including rodent bocavirus and rat bocavirus. Recently, ungulate bocaparvovirus 4, a known porcine bocavirus, has also been found in rats. Thus, investigating bocaviruses in rats is important for determining the origin of the viruses and preventing and controlling their transmission. To the best of our knowledge, no study to date has investigated bocaviruses in the livers of rats. In this report, a total of 624 rats were trapped in southern China between 2014 and 2017. Liver and serum samples from rats were tested for the prevalence of bocaviruses using PCR. Sequences related to ungulate bocaparvovirus 4 and rodent bocavirus were detected in both liver and serum samples. Interestingly, the prevalence of ungulate bocaparvovirus 4 (reference strain:KJ622366.1) was higher than that of rodent bocavirus (reference strain:KY927868.1) in both liver (2.24% and 0.64%, respectively) and serum samples (2.19% and 0.44%, respectively). The NS1 regions of ungulate bocaparvovirus 4 and rodent bocavirus related sequences displayed over 84% and 88% identity at the nucleic acid and amino acid levels, respectively. Furthermore, these sequences had similar genomic structure, genomic features, and codon usage bias, and shared a common ancestor. These viruses also displayed greater adaptability to rats than pigs. Our results suggested that ungulate bocaparvovirus 4 and rodent bocavirus may originate from rats and may be different genotypes of the same bocavirus species.     

Structure of the NS1 Protein N-Terminal Origin Recognition/Nickase Domain from the Emerging Human Bocavirus

Human bocavirus is a newly identified, globally prevalent, parvovirus that is associated with respiratory infection in infants and young children. Parvoviruses encode a large nonstructural protein 1 (NS1) that is essential for replication of the viral single-stranded DNA genome and DNA packaging and may play versatile roles in virus-host interactions. Here, we report the structure of the human bocavirus NS1 N-terminal domain, the first for any autonomous parvovirus. The structure shows an overall fold that is canonical to the histidine-hydrophobic-histidine superfamily of nucleases, which integrates two distinct DNA-binding sites: (i) a positively charged region mediated by a surface hairpin (residues 190 to 198) that is responsible for recognition of the viral origin of replication of the double-stranded DNA nature and (ii) the nickase active site that binds to the single-stranded DNA substrate for site-specific cleavage. The structure reveals an acidic-residue-rich subdomain that is present in bocavirus NS1 proteins but not in the NS1 orthologs in erythrovirus or dependovirus, which may mediate bocavirus-specific interaction with DNA or potential host factors. These results provide insights into recognition of the origin of replication and nicking of DNA during bocavirus genome replication. Mapping of variable amino acid residues of NS1s from four human bocavirus species onto the structure shows a scattered pattern, but the origin recognition site and the nuclease active site are invariable, suggesting potential targets for antivirals against this clade of highly diverse human viruses.     

The Human Bocavirus Is Associated with Some Lung and Colorectal Cancers and Persists in Solid Tumors

Human bocavirus is the second autonomous human parvovirus with assumed pathogenic potential. Other parvoviruses are known to persist and even integrate into the host genome, eventually contributing to the multi-step development of cancer. Human bocavirus also persists in an unknown percentage of clinically asymptomatic patients in addition to those with primary infection. The aim of the present study was to analyze the role of Human bocavirus in lung and colorectal cancers. Therefore, formalin-fixed, paraffin-embedded, archived tumor samples were screened for Human bocavirus DNA by PCR, Southern blotting, and sequencing. Positive tissues were further subjected to fluorescence in situ hybridization analysis to specifically detect human bocavirus DNA in the infected cells. In total, 11 of the 60 (18.3%) lung and 9 of the 44 (20.5%) colorectal tumors tested positive for human bocavirus DNA by PCR and were confirmed by sequencing and fluorescence in situ hybridization analysis. Thus, human bocavirus DNA is present in the nuclei of infected cells, in either single or multiple copies, and appears to form concatemers. The occurrence of these human bocavirus DNA structures supports the existence of the postulated σ- or rolling-hairpin replication mechanism. Moreover, the fluorescence in situ hybridization patterns inspired the hypothesis that human bocavirus DNA either persists as cccDNA or is integrated into the host genome. This finding suggests that this virus may indirectly contribute to the development of some colorectal and lung cancers, as do other DNA viruses, such as the human hepatitis B virus, or may play an active role in cancer by interacting with the host genome.     

Bocavirus episome in infected human tissue contains non-identical termini

Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus [1] in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses. Significance: HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.     

Genomic characterization and high prevalence of bocaviruses in swine

Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ～2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45-75% and 55-70% respectively, with 30-47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses.     

可调控表达人博卡病毒1型非结构蛋白NS1稳定细胞系的建立及其反式转录激活作用初探

人博卡病毒1型(Human bocavirus 1,HBoV1)非结构蛋白NS1是多功能蛋白,对病毒复制有重要作用,同时可诱导宿主细胞凋亡。在研究NS1蛋白功能时,降低NS1蛋白对宿主细胞的毒性作用是急需解决的问题。基于此,文中建立了可调控表达HBoV1非结构蛋白NS1的稳定细胞系。构建NS1重组慢病毒质粒(含可调控启动子),应用转染试剂将NS1重组慢病毒质粒转染至HEK293T细胞。通过嘌呤霉素筛选抗性细胞、多西环素诱导NS1表达,建立可稳定表达NS1-100、NS1-70蛋白的HEK 293T细胞系,利用荧光标记蛋白和Western blotting检测,确定NS1蛋白的表达。并在稳定表达NS1细胞系中转染HBoV1启动子-荧光素酶基因的质粒,分析NS1的反式转录激活活性。结果表明NS1蛋白可在建立的细胞系中稳定表达,且稳定表达NS1蛋白对HBoV1启动子有较强的激活活性,为进一步研究非结构蛋白NS1的功能及人博卡病毒致病机理奠定了良好的基础。     

猪博卡病毒环状附加体的发现与鉴定

为了证实博卡病毒可以环状附加体形式存在于宿主体内,本研究利用半巢式PCR方法在健康猪粪便标本中筛查出2株猪博卡病毒环状附加体PBoV_G2-episome和PBoV_G3-episome。通过反复测序和序列拼接得到其末端非编码区序列（405nt和511nt）。经过对其进行序列分析及二级结构的预测,发现PBoV_G2-episome与人博卡病毒3附加体（HBoV3-episome）结构相似,而PBoV_G3-episome与博卡病毒属其他成员的末端二级结构存在较大差别。猪博卡病毒环状附加体的发现证实了有些博卡病毒与其他细小病毒的复制方式存在一定差异,也为今后博卡病毒感染性克隆的构建提供了一条研究思路。     

Sequence analysis of an isolate of minute virus of canines in China reveals the closed association with bocavirus

In the present study, we have cloned and sequenced the nearly-full-length genome of minute virus of canines (MVC), SH26, in China. The genome of MVC, 5,132 nucleotides (nts) in length, contains three open reading frames (ORFs), which are 2,325-bp of NS1, 561-bp of NP1 and 2,112-bp of VP1/VP2 encoding three proteins of 774, 186 and 703 residues, respectively. Predicted amino acids sequence of NS1 of MVC has 44% identity with human bocavirus (HBoV) and human boacvirus 2 (HBoV2), NP1 has 48 and 45% identity with HBoV and HBoV2, VP1/VP2 has 45 and 46% identity with HBoV and HBoV2, respectively. Phylogenetic analysis showed that the present Chinese MVC strain was also closely clustered with the previous American and Japanese MVC isolates, and MVCs formed a different branch together with bovine parvovirus and HBoVs from other parvoviruses classified into Parvovirinae.     

The human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification

The major objective of this study was to investigate the pathogenic role of human bocavirus (hBoV) in patients hospitalized with acute respiratory tract infection (ARTI). Overall, 685 respiratory samples from 426 patients were examined by PCR for human bocavirus, as well as for other known human respiratory viruses. HBoV was quantified by PCR. Forty/283 (14.1%) pediatric patients, and 2/143 (1.4%) adult patients were found to harbor hBoV for a total of 45 episodes (16 detected as single infection, and 29 as coinfection) of hBoV-associated respiratory infection. HBoV DNA quantification revealed the presence of an NPA viral load > 1.0 x 10(5) DNA copies/ml in respiratory secretions from 17/40 (42.5%) children and 0/2 adults. Below this cut-off, hBoV appeared to be an innocent bystander or a persistent virus. Although hBoV may be frequently detected in children with upper or lower ARTI, in less than 50% young patients it appears to be potentially pathogenic.