摘要
目的了解粤东地区喘息性疾病患儿的病原体中是否存在人类偏肺病毒(Human Metapneumovirus,HMPV)并分析其N蛋白编码基因的特征,同时探讨其感染的临床特征,为临床防治小儿喘息性疾病提供病原学基础实验依据。
材料与方法
材料:选取2006年6月1日至2007年6月1日期间因喘息性疾病收住院治疗的2岁以下患儿(240例),采集其鼻咽吸取物或咽试子以供病毒学检测之用,并收集其年龄、性别、症状、体征等临床资料以供分析研究;同时采集同时期健康体检儿童(100例)的咽试子作为对照研究。
方法:1.应用多重PCR筛查呼吸道合胞病毒(RSV),腺病毒(ADV),流感病毒A型,B型(IVA,B),副流感病毒1型,3型(PIV1,3) ,鼻病毒(RV),博卡病毒( HBOV)。
2.应用针对HMPV的N蛋白基因的RT-PCR引物进行HMPV基因片段检测,随机选取6份RT-PCR扩增阳性产物进行核苷酸序列测定。将所测序列与GenBank中公布的基因序列进行比较分析,通过生物信息学方法对测定的人偏肺病毒基因片段进行进化分析,分析与其它地方病毒株的同源性。同时对HMPV N蛋白基因阳性病例的临床资料进行分析。
结果
1.240份小儿喘息性疾病呼吸道标本中,病毒阳性101份(42%),单一病毒感染86份(85%),混合感染15例(占15%);检出病毒中以RSV占首位,41例(占41%),其次递减为PIV3 18例(占18%),RV 17例(占17%), IVA 16例(占16%),PIV1 13例(占13%),HMPV 12例(占12%),ADV 5例(占5%), HBOV 4例(占4%),未检测到IVB。
2.240份标本中HMPV阳性率为5%(12/240),在其他病毒阴性标本中阳性率为7.9%(其中2例与其他病毒混合感染,分别与ADV和RV),其中3、4月份为发病高峰。阳性患儿均为年龄较小的患儿。6份HMPV阳性标本目标基因部分核苷酸序列与GenBank中公布的多株HMPV N基因同源性为80.8%～98.4%。核苷酸序列基因进化树分析显示存在2种不同的基因型。12例HMPV阳性患儿均表现发热、咳嗽及喘息等临床症状;临床诊断为毛细支气管炎8例、喘息型肺炎4例。
3.100份健康体检儿童的呼吸道标本中均未检测到HMPV特异性基因片段;检出PIV3:1例,RSV:3例,RV:4例,其余病毒未检出。
结论HMPV是粤东地区婴幼儿喘息性疾病的重要病毒性病原体之一;与常见呼吸道病毒感染所致疾病的临床表现相似;粤东地区儿童感染的HMPV株同时存在2种不同的基因型。
Background and objective To analyze the status of the human metapneumovirus(HMPV) in wheezing children in East Guangdong and the clinical characteristic of the diseases with HMPV infection. To anlyze the sequence of the N genes of HMPV.
Material and methods
Material: From June 1,2006 to June 1,2007,a total of 340 nasopharyngeal secretions or throat swab samples(240 from children with wheezing, 100 from health children) were collected from children with wheezing.
Methods: 1. respiratory syncytial virus (RSV), human bocavirus(HBOV),influenza virus type A, B(IV A, B), Rhinovirus(RV),parainfluenzavirus type 1,3 (PIV1, 3) and adenovirus(ADV) were investigated by multiplex polymerase chain reaction (PCR). 2. The viral nucleic acid of HMPV N gene of 323 samples were amplified by RT-PCR, and six HMPV N gene positive PCR products were randomly chosen for purified and sequenced. Nucleotide sequences alignment and phylogenetic analysis were performed with DNAstar software. The clinical data of the children with HMPV infection were analyzed.
Results
1.Viruses were detected in 101 of all cases(42%), with RSV in 41(41%) the most frequent,followed PIV3 in 18(18%), RV in17 (17%), IV A in 16(16%), PIV1 in13 (13%),HMPV in 12(12%), ADV in 5(5%), HBOV in 4(4%)and. Mixed viral infection was detected in 15 cases.
2.The positive rate of the HMPV infection identified from 240 children with wheezing was 5%(12/240). The positive rate of HMPV from the negative samples of other virus was 7.9%. HMPV was significantly more prevalent in March and April(5/12). Six HMPV N gene fragments of sequence were closely related to HMPV in the GenBnk. Similarity of HMPV partial N gene with HMPV N gene published at nucleotide levels varied from 80.8 to 98.4. Phylogenetic tree analysis of nucleotide sequences revealed 2 different genotypes. All 12 HMPV-posotive patients suffered from fever, cough and wheeze. The clinical diagnoses of the HMPV-posotive patients were wheezy pneumonia (four patients), bronchiolitis(eight patients).
3.None of the throat swabs 100 cases from healthy subjects tested HMPV-positive. 1 PIV3, 3 RSV and 4 RV had detected, None of other virus had detected.
Conclusions HMPV is an important viral pathogen in children with wheezing in East Guangdong, however its clinical signs were not specific. HMPVs of different genotypes may co-circulate in children in East Guangdong.
引文
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