A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses

The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT‐PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV‐A, 40 HEV‐B, and 10 HEV‐C) tested, 100% concordance in HEV‐A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV‐B and HEV‐C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV‐A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV‐A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease. J. Med. Virol. 82:649–657, 2010. © 2010 Wiley‐Liss, Inc.     

人白细胞抗原-DPB1基因直接测序分型技术

研究建立准确的基于人白细胞抗原一ＤＰＢ１（ＨＬＡ－ＤＰＢ１）核酸序列的基因分型技术,为疾病与ＨＬＡ基因相关性研究及器官、组织移植提供准确的ＨＬＡ基因分型方法。使用第十一届国际组织相容性会议提供的引物,经ＰＣＲ技术扩增、分离相应的基因片段,纯化后用ＰＥ公司四色荧光染料终止剂标记循环测序技术直接测定核酸序列,获得个体基因型序列资料,通过与基同型资料数据库比较确定基因型别。这一技术可以准确地确定个体的 ＨＬＡ－ＤＰＢ１的基因型别并得到核酸序列。为进一步研究奠定了基础。本技术可用于其它类似的研究工作。     

Poliovirus serotype-specific VP1 sequencing primers

The Global Polio Laboratory Network routinely uses poliovirus-specific PCR primers and probes to determine the serotype and genotype of poliovirus isolates obtained as part of global poliovirus surveillance. To provide detailed molecular epidemiologic information, poliovirus isolates are further characterized by sequencing the ~900-nucleotide region encoding the major capsid protein, VP1. It is difficult to obtain quality sequence information when clinical or environmental samples contain poliovirus mixtures. As an alternative to conventional methods for resolving poliovirus mixtures, sets of serotype-specific primers were developed for amplifying and sequencing the VP1 regions of individual components of mixed populations of vaccine-vaccine, vaccine-wild, and wild-wild polioviruses.     

Molecular characterization of human enteroviruses in clinical samples: comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.     

Typing of human papillomavirus by pyrosequencing

The possibility of using a new bioluminometric DNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.     

Molecular detection and characterization of human enteroviruses directly from clinical samples using RT-PCR and DNA sequencing

Enteroviruses are common human pathogens associated with a wide spectrum of symptoms ranging from asymptomatic infection to acute flaccid paralysis and neonatal multi-organ failure. Molecular methods that provide rapid diagnosis and increased sensitivity have been developed for the diagnosis of enterovirus infection using oligonucleotide primers complementary to conserved sequences located in the 5' untranslated region (UTR), but data generated from these regions are not sufficiently discriminatory for typing due to the lack of correlation between their nucleic acid sequence and serotype specificity. Sequences derived from the gene encoding the capsid VP1 correlate with serotype, and therefore provide the opportunity for the development of molecular typing methods consistent with present serogical methods. In this study, oligonucleotide primers that amplify a region of the 5'UTR to detect enterovirus RNA, and the region encoding the enterovirus VP1 N-terminus to characterize virus strains were used in nested and semi-nested RT-PCRs, respectively. The ability of the VP1 RT-PCR to amplify diverse viruses within genotypes and genogroups was confirmed by the correct identification of both prototype strains, and strains circulating currently of the same genotypes. The molecular methods proved their utility through the detection of enteroviruses that failed to grow in cell culture, their subsequent characterization and the characterization of strains that failed to serotype in neutralization assays. Molecular methods increased significantly the sensitivity of detection (P < 0.001) and of characterization (P < 0.01) of enteroviruses when compared to classical methods.     

Receptor specificity of human enteroviruses

The review presents the currently available data on the receptor specificity of enteroviruses. It discusses whether changes in the receptor specificity of enteroviruses may play a role in their in vitro and in vivo reproduction.     

鸡贫血病毒VP2基因的克隆、序列分析及表达

目的：为探讨鸡贫血病毒的分子生物学特征。方法：采用ＰＣＲ方法获得允许贫血病毒的ＶＰ２基因,采用平端连接将其克隆到ＰＵＣ１１９载体上进行测序,采用粘端连接将其亚克隆到ＰＥＴ载体上并进行原核表达。结果：发现ＣＡＶ－ＳＪ１株的ＶＰ２基因共有６５１个碱基,同围外ＴＫ５８０３,２６Ｐ４,Ｃｕｓ－１、８２０２毒株的ＶＰ２基因相比分别有６、７、８、７个碱基的差别,其中３个是ＳＪ１株特有的碱基变化。由基因序列推导     

Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene

Summary. Human adenoviruses (Ads) are responsible for a substantial disease burden. Type-specific identification of Ads can help guide therapeutic and disease prevention strategies and aid epidemiological investigations. Immunotyping of Ads by serum neutralization (SN) is laborious and time consuming and depends upon type-specific antisera that are in short supply. A rapid molecular typing assay based on polymerase chain reaction (PCR) amplification and sequencing of Ad hexon gene hyper-variable regions 1–6 (HVR_1–6) known to contain type-specific epitopes was evaluated as an alternative to SN. Deduced amino acid sequences of HVR_1–6 obtained from all 51 currently recognized Ad prototype strains were well resolved, with the exception of types 15 and 29, which were identical. Of 192 temporally and geographically diverse Ad field isolates sequenced in this study, and 111 previously published sequences, all more closely matched their predicted prototype strains. Ads were also detected and correctly identified directly from 24 clinical specimens positive by culture or antigen detection. PCR and sequencing of HVR_1–6 offers a practical alternative to SN for typing most Ads and can be readily adapted for use in laboratories with molecular capabilities.     

人类肠道病毒的研究进展

自 1908年脊髓灰质炎病毒被鉴定以来,已发现超百种肠道病毒致病血清型,可引起手足口病、新生儿败血症样疾病、无菌性脑炎、急性弛缓性脊髓炎、呼吸系统疾病、急性出血性结膜炎等多种疾病。近年来,诸如 EV-A71 和 EVD68等肠道病毒趋于全球化周期性流行,已成为公共卫生领域不可忽视的重要挑战之一。然而,当前针对肠道病毒的防控手段仍然非常有限,迫切需要深入探究肠道病毒致病的分子机制,为开发高效、安全、广谱的抗肠道病毒药物提供新靶点与新思路。本文通过对肠道病毒的分类、流行病学、结构、生命周期、病毒蛋白与宿主因子相互作用、并发症致病机制与临床治疗以及动物感染模型的确立与应用等方面进行系统的综述,旨在为肠道病毒防治提供理论参考依据,为肠道病毒的药物研发与疫苗储备库建设提供新的思路。     

地表水中肠道病毒检测结果分析

目的了解某地地表水中EV71、CoxA16等人肠道病毒的污染情况。方法选择某地1个污水处理厂和1条河流（上、下游）作为采样对象,每月采集未经处理的污水和上、下游河水各1份,经过滤、浓缩后进行人肠道病毒核酸检测。结果 36份水样中,肠道病毒检出率为52.77%;其中EV71阳性检出率为44.44%,CoxA16阳性检出率为16.66%,其它肠道病毒的检出率亦高达36.11%。夏季地表水体中的肠道病毒阳性率（100.00%）显著高于其它季节（37.03%）（P〈0.00）。污水中各类肠道病毒的检出率与河水中肠道病毒的检出率差异无统计学意义（P〉0.05）。地表水中肠道病毒的检出率与当地手足口病、手足口病重症及肠道传染病（甲乙丙类）的发生无相关（P〉0.05）。结论地表水中肠道病毒的污染严重,有必要开展进一步的研究了解肠道病毒经水传播的风险。     

Presumed common source outbreaks of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region

Hepatitis A virus isolates from anti-HAV IgM positive sera of 70 hepatitis cases in two outbreaks and 216 other cases in Central America, 136 sporadic cases and 53 cases from an hyper-endemic region in Costa Rica, were compared by phylogenetic analyses within the VP1 region. The outbreaks in all 531 cases, in 1992 and 1999, respectively, were presumed water borne. In the first outbreak, HAV RNA could be detected in 70% of the cases sampled during 6 weeks after onset of jaundice. In the hyper-endemic region of San Ramón in Costa Rica, 1,932 cases were registered between 1972 and 1985. All isolates belonged to subtype 1A. Background isolates from Costa Rica and El Salvador tended to form separate subclusters in the phylogenetic tree construction and were mostly unrelated to subtype 1A strains from other parts of the world. Based on their amino acid sequences, four HAV strains, all related to CR326 sampled in Costa Rica in 1960, were found to have circulated in the area during the last three decades. However, on the basis of nucleotide variability the isolates from the outbreaks could be distinguished from the strains from sporadic cases and sequence analysis could confirm the epidemiological homogeneity of both outbreaks. In the hyper-endemic region, 16 different sequences were encountered forming one single subcluster. Thus, limited sequencing within the VP1 region proved useful to identify outbreaks of hepatitis A in a highly endemic area, where most strains were local and only one subtype was prevalent.     

Detection by PCR of human polyomaviruses BK and JC in immunocompromised individuals and partial sequencing of control regions

Immunocompromised individuals were tested for the presence of the human polyomaviruses JC (JCV) and BK (BKV) by the polymerase chain reaction (PCR). The use of appropriate primers in a nested PCR allowed the detection of both viruses simultaneously. Viruses were differentiated by restriction fragment length analysis of amplified DNA fragments. Both BKV and JCV DNA were detected in the urine of an AIDS patient with progressive multifocal leukencepha-lopathy. In autopsy materials from this patient, JCV- but not BKV-DNA was found in brain and kidney tissue, whereas lung tissue was negative for both virus DNAs. To evaluate the methology further, hybridization-positive urines from three recipients of bone marrow transplants and a positive urine of an acute myeloid leukemia patient were analyzed by this PCR method. One case was positive both for BKV and JCV, two cases were positive only for BKV, and one was negative for both. Parts of the control regions of JCV and BKV were sequenced directly from PCR-derived fragments. The JCV sequence from urine of the AIDS patient compared to sequences from a bone marrow transplant recipient and to archetypical reference strains showed two nucleotide (nt) exchanges out of 250 nt. The BKV sequences from the AML and the AIDS patients showed five nt exchanges out of 265 nt in the control region and were identified as BKV WW or WWT3 strains. In the agnogene region five exchanges were detected, two of them resulting in non-conservative amino acid exchanges. The possibility of testing clinical specimens of different origins by this PCR method is important for elucidating often unclear clinical courses in immunocompromised patients. Furthermore, our results show the versatility of PCR for diagnosis and for molecular characterization of human polyomaviruses.     

北京地区新近报道的人Boca病毒VP1、VP2蛋白编码区基因序列分析

目的了解北京地区新近报道的人Boca病毒（human bocavirus，HBoV）主要结构蛋白编码区基因的特征。方法选择已经过初步研究证明为HBoV NP1基因检测为阳性的2份临床标本BJ3064、BJ3722。应用针对HBoV VP1蛋白编码区基因的PCR引物进行扩增，对所获得的PCR扩增产物直接进行核苷酸序列测定。将所测到的序列与GenBank中的基因序列进行比较分析和种系进化分析。结果从标本BJ3064及BJ3722中扩增得到HBoV VP1蛋白编码区全基因的PCR扩增产物为2016bp，编码671个氨基酸。VP2蛋白是在不改变开放性读码框架（ORF）的情况下，由VP1蛋白编码区内起始合成，并与VP1终止于同一终止密码子，长度为1629bp，编码542个氨基酸。与HBoV原型株ST1、ST2株相比较，BJ3064、BJ3722的VP1及VP2蛋白无论是核苷酸水平还是氨基酸水平的同源性均超过98％，但与同属细小病毒的BPV及MVC相应位置的序列相比较，同源性较低，其中核苷酸序列同源性低于60％，而氨基酸序列同源性低于50％。VP1及VP2蛋白的编码区基因进化分析显示。BJ3064、BJ3722与ST2之间进化关系较ST1更密切。在BJ3064、BJ3722的VP1蛋白中，也存在类似于MVC的保守性磷酸酯酶A2特异性位点的活性基序（HDXXY）及Ca^2＋结合位点。结论已得到HBoV的结构蛋白VP1和VP2的全基因，将为儿科急性呼吸道感染中该病毒的病原作用、地位及其在各年龄组人群中的血清学特征的深入研究打下坚实的基础。