Edinburgh Research Explorer Epidemiology and clinical associations of human parechovirus respiratory infections
Edinburgh Research Explorer Epidemiology and clinical associations of human parechovirus respiratory infections
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人类副病毒(HPev)感染在幼儿中普遍存在，并与轻度胃肠炎有关，较少与脑膜炎和新生儿败血症有关。为了调查这些病毒与呼吸道疾病的关系，采用高灵敏度的套式聚合酶链式反应对2007年1月至12月期间收集的大量呼吸道标本进行了筛选。此前，通过聚合酶链式反应对呼吸道样本进行了八种呼吸道病毒的检测，包括呼吸道合胞病毒和腺病毒。3844份样本中有34份检出HPeV，占2220份研究对象中的27份(1.2%)。用fi直接从临床标本中扩增出VP3/VP1区fi，通过测序鉴定HPeV型。该方法可扩增出所有高敏感性的HPeV型(1型和3型至6型)，并可在除1例筛查阳性研究标本(25例HPeV1和8例HPeV6)外的所有标本中鉴定出fi。HPeV1和HPeV6的感染都是季节性的，以20(CID：3)L反应体积的厂家提供的缓冲液(含0.4 mM脱氧核苷三磷酸和2.4 mM硫酸镁)、6(CID：3)L提取的RNA、0.8(CID：3)上标III RT/白金Taq聚合酶的L和0.5(CID：3)M的外部VP3/VP1引物进行RT-PCR20(CID：3)检测频率最高。第一轮fi的反转录和聚合酶链式反应循环条件依次为43℃1 h，53℃(1分钟)和55℃(1分钟)20个循环，然后70℃15分钟和94℃2分钟。聚合酶链式反应包括94°C(S 30个)、50°C(S 30个)和68°C(10 5个S)的40个循环，并在68°C下fiNAL延伸5min。将一微升的产物转移用于第二次聚合酶链式反应，按照5(Cid：2)非编码区聚合酶链式反应的描述进行，但使用VP3/VP1内引子。用琼脂糖凝胶电泳法显示304bP的fi-NAL产物。使用BigDye终止器试剂盒(英国沃灵顿的应用生物系统公司)在两个方向上直接对扩增的fi进行测序，从该试剂盒组装第2182和2437位之间的完整序列。类型相同的fi阳离子是通过使用包含先前指定的HPeV型的已发表的HPeV变异体序列的数据集对核苷酸和推断的氨基酸序列进行系统发育分析来实现的。VP3/VP1区核苷酸GenBank FJ009260 FJ009290。
Infections with human parechoviruses (HPeVs) are prevalent in young children and have been associated with mild gastroenteritis and, less frequently, with meningitis and neonatal sepsis. To investigate the involvement of these viruses in respiratory disease, a highly sensitive nested PCR was used to screen a large archive of respiratory specimens, collected between January and December 2007. Respiratory samples had previously been tested for eight respiratory viruses, including respiratory syncytial virus and adenovirus, by PCR. HPeV was detected in 34 of 3,844 specimens, representing 27 of 2,220 study subjects (1.2%). HPeV types were identified by sequencing the VP3/VP1 junction amplified by PCR directly from clinical specimens. The assay could amplify all HPeV types examined with high sensitivity (types 1 and 3 to 6) and also identified HPeV types in all but one of the screen-positive study specimens (25 HPeV1 and eight HPeV6 specimens). Infections with both HPeV1 and HPeV6 were seasonal, with highest frequencies in Each combined RT-PCR was carried out in a 20- (cid:3) l reaction volume of the manufacturers’ supplied buffer (containing 0.4 mM deoxynucleoside triphosphates and 2.4 mM MgSO 4 ), 6 (cid:3) l of extracted RNA, 0.8 (cid:3) l of Superscript III RT/Platinum Taq polymerase, and 0.5 (cid:3) M of outer VP3/VP1 primers. The RT and PCR cycling conditions for the first round were, sequentially, 43°C for 1 h and 20 cycles of 53°C (1 min) and 55°C (1 min), followed by 70°C for 15 min and 94°C for 2 min. PCR comprised 40 cycles of 94°C (30 s), 50°C (30 s), and 68°C (105 s) and a final extension at 68°C for 5 min. One microliter of the product was transferred for a second PCR, which was performed as described for the 5 (cid:2) UTR PCR but with the VP3/VP1 inner primers. The final product of 304 bp was visualized by agarose gel electrophoresis as described above. Amplified DNA was directly sequenced using a BigDye Terminator kit (Ap-plied Biosystems, Warrington, United Kingdom) in both directions, from which a complete sequence between positions 2182 and 2437 was assembled. Type identification was achieved by phylogenetic analysis of nucleotide and inferred amino acid sequences using a data set containing sequences from published HPeV variants of previously assigned HPeV types. Nucleotide the VP3/ VP1 region GenBank FJ009260 FJ009290.