北京地区急性呼吸道感染婴幼儿中人副流感病毒感染状况的研究

Human parainfluenza virus infections in infants and young children with acute respiratory infections in Beijing

目的了解北京地区急性呼吸道感染患儿中人副流感病毒(HPIV)的感染状况。方法(1)根据 HPIV 血凝素蛋白(HA)的基因保守区序列,合成能够区分 HPIV 1～4型的多重巢式 PCR 引物,并用多种呼吸道病毒的标准毒株确定多重 PCR 方法的特异性;(2)收集2003年8月-2006年4月急性呼吸道感染患儿的临床标本3519例,应用逆转录多重 PCR 方法,对标本同时进行 HPIV 1～4的检测。(3)随机选取不同型别的 HPIV 基因检测阳性标本共10例,对其 PCR 扩增产物直接进行核苷酸序列测定,并将所测到的序列与 GenBank 中的基因序列进行比较。结果 (1)多重 PCR 方法的特异性检测显示,HPIV 的分离株 cDNA 有预期大小的扩增片段,与其他常见呼吸道病毒无交叉反应性。(2)3519例标本中多重 PCR 检测阳性的为349例,占本组检测标本的9.9%(349/3519),明显高于间接免疫荧光方法和(或)病毒分离4.8%的阳性率(170/3519);应用多重 PCR 方法还检测到HPIV1与 HPIV3以及 HPIV3与 HPIV4的混合感染;在上呼吸道及下呼吸道感染的患儿中均有较高的HPIV 阳性检出率;季节性分布分析显示,HPIV 感染无明显的规律性,以 HPIV1、3为主,HPIV2、4仅散发存在。(3)序列分析结果提示,所选取的多重 PCR 方法检测阳性的10例标本确实是 HPIV 阳性,其中有两份分别属于 HPIV 4A、4B 亚型。结论 HPIV 在儿童感染中除了单-亚型感染外还可存在混合型感染。未发现其感染的季节性特点。所应用的逆转录多重 RT-PCR 方法不仅检出率高于常用的方法(间接免疫荧光方法和病毒分离),而且能够直接进行亚型分析。除了检出常见的1、2、3型外,还检出了较少见的4型,属国内首次报道。

Objective To understand the impact of human parainfluenza virus （HPIV） on acute respiratory infections in infants and young children in Beijing. Methods Multiplex reverse transcription- PCR was used to amplify the hemagglutinin （HA） gene fragment of HPIV from clinical specimens. Primer pairs derived from a conserved region of the HA genes of HPIV were used to develop the multiplex RT-PCR for detecting and typing HPIV. The sensitivity and specificity of the method were determined by using various RNA and DNA viruses as controls. Specimens collected from 3519 children with acute respiratory infections from Aug. 2003 to Apr. 2006 were analyzed for HPIV by the multiplex RT-PCR as well as for other respiratory viruses by virus isolation and/or indirect immunofluorescent assay （IFA）. Ten amplicons with expected molecular weight matching different types of HPIV were randomly selected for sequence analysis. Results Only the cDNA from the isolated strains of HPIV 1 and 3 was positive by the multiplex RT-PCR. Phylogenetic analysis for those 10 amplicons＇ sequences which belong to HPIV 1 - 4 types respectively as determined by multiplex-PCR indicated that these specimens were truly HPIV positive. These 10 HPIV positive specimens included two specimens of type 4 which was further subtyped as HPIV4A and 4B by sequence analysis. With the multiplex RT-PCR, HPIV were detected in 349 out of 3519 specimens with the positive rate of 9.9% （349/3519）, which is higher than 4.8% by the methods of virus isolation and/or IFA. And the HPIV positive rates were high in patients with not only acute upper but also lower resviratory tract infection. No regular seasonality distribution of HPIV infection was found. HPIV 1 and 3 were more common than HPIV 2 and 4. Conclusion With higher sensitivity and specificity than virus isolation and IFA, multiplex RT-PCR is beneficial for the etiologic and epidemiologic studies on HPIV, as well as for HPIV typing. The data from this study indicate that HPIV is one of the important etiological viruses of acute respiratory tract infections in infants and young children in Beijing,