摘要
手足口病(Hand, Foot and Mouth Disease, HFMD)是一组由人肠道病毒(Human Enterovirus, HEV)引起的急性传染病,临床症状以发热和手、足、口腔和臀部等部位出现皮疹为主,绝大部分患者预后良好,一般可在发病后5-7日内自愈。但有的时候会出现比较严重的并发症,包括心肺并发症(如急性神经源性肺水肿、肺出血和心肌炎等)和神经系统并发症(如脑干脑炎、无菌性脑膜炎和急性弛缓性麻痹等),严重时可引起死亡。
HFMD传染性极强,易导致流行或暴发,为一种全球性常见传染病,世界上有许多国家都报道了数起大规模的HFMD的暴发,特别是近几年来,东亚和东南亚的一些国家都暴发了大规模的疫情,如新加坡、韩国、马来西亚、日本、越南以及中国,在这些暴发中,均有患儿死亡的报道。其中2008年中国安徽阜阳发生HFMD暴发流行,3月1日至5月9日共报告病例6049例,死亡22例,HFMD成为了我国重大的公共卫生事件,国家卫生部于2008年5月2日,将手足口病列入传染病防治法规定的丙类传染病进行管理。而在2008年以后,HFMD在我国一直持续大流行,并且不断有重症和死亡病例的报告。
为了应对HFMD疫情,我们选取河南省洛阳市、开封市,在同一时间范围内采集临床诊断为HFMD病例标本,进行HFMD致病原的检测和研究,分析两城市致病病原谱和致病原的基因特征,构建相关病原的系统发生树,并通过与国内外不同病原比较来研究我国HFMD病原谱的特征,为建立我国HFMD病原谱提供基线资料,从而为HFMD疫苗的研发,临床诊断以及预防控制提供参考和科学依据。
在对2009年河南省洛阳市和开封市HFMD疫情简要分析时发现,2009年HFMD在两市全年均有发生,高峰期集中在3-6月,有别于以前报道的在春夏季暴发流行的特点,表明HFMD已经突破了季节性这一瓶颈,转变成全年均可发生的传染病。
本研究选取两市HFMD流行高峰采集发病7日内患者标本进行实验研究,共采集粪便标本327份。在对粪便标本进行处理后选用人横纹肌肉瘤细胞(RD细胞)、人喉癌上皮细胞(HEp-2细胞)进行病毒分离,在该实验环节中,引入了核酸杂交技术,即在标本进行盲传二代后,对病毒分离呈阴性的标本进行HEV核酸杂交实验,对杂交阳性样品再进行三代培养。结果显示,在病毒分离培养工作中引入该技术,可较明显地提高病毒分离率,本研究中在RD细胞和HEp-2细胞中分别提高了8.57%和0.62%,最终获得RD细胞病变230份,HEp-2细胞病变8份。
随后采用了RT-PCR结合核苷酸序列测定的方法获得病毒的部分核苷酸序列,再对所获得的核苷酸序列通过美国国家生物技术信息中心BLAST程序中的一致性评分来判定核苷酸序列的同源性,最终确定病毒株型别,从而获得河南省洛阳市和开封市的HFMD病原谱。
本研究共获得河南省洛阳市HEV毒株152株,其中118株人肠道病毒71型(Human enterovirus71, HEV71),26株柯萨奇病毒A组16型(Coxasckievirus A16, CVA16),3株CVA6、CVA2、CVA4、 CVB2、CVA24、埃可病毒30型(echovirus30, ECHO30)各1株；获得河南省开封市HEV毒株82株,鉴定HEV7166株,CVA1610株,CVA24、CVB5各2株,ECHO7、CVA4各1株,另外获得人腺病毒(Human Adeno virus, HAdV)3株。
排除了合并感染和非HEV对HFMD病原确定的干扰后,最终确定2009年河南省洛阳市HFMD的病原谱为HEV71(78.76%)、CVA16(17.33%)、CVA6(2.00%)、CVA4(0.67%)、CVA2(0.67%)、CVB2(0.67%),2009年河南省开封市HFMD的病原谱为HEV7(81:48%)、 CVA16(12.35%)、CVA4(1.23%)、CVB5(1.23%)、ECHO7(1.23%)、 CVA24(1.23%)。
利用BioEdit和MEGA4.0软件对以上病毒株和从Genbank数据库上下载的各型别病毒代表株进行了基因亲缘性关系分析、核苷酸和氨基酸的同源性分析。
对HEV71分析显示,河南省洛阳市和开封市的病毒株均属于C4基因亚型的C4a进化分支,洛阳市HEV71至少存在4个传播链,而开封市至少存在2个传播链,均以各自第1传播链为优势传播链。两地的病毒株在核苷酸和氨基酸序列上均有很高的同源性,具有各自不同的地域特点,它们同我国其它地区的HEV71保持着相同的进化方向。
CVA16可分为2个基因型,B基因型又可以分为B1和B2两个基因亚型,B1基因亚型可进一步分为B1a和B1b进化分支,而分离自洛阳市和开封市病例的CVA16在两个进化分支均有分布,但分布在各自进化分支上的病毒株数差别很大。两地也存在多个传播链同期在流行的现象。同时发现,两地的病毒株在核苷酸同源性并不算很高的情况下,氨基酸却保持很高的同源性,这种现象很值得我们去进一步关注。
基于CVA4VP1区全长核苷酸序列,在可获得VP1核苷酸序列有限的情况下,经过计算和分析,认为可将CVA4分为5个基因型,即A～E。各基因型间VP1核苷酸序列均存在15%以上的平均差异,而在系统发生树中,分离自洛阳市和开封市的CVA4病毒株同处于C基因型,且它们聚集成簇,保持着很高的核苷酸和氨基酸同源性,表明虽然两地相距甚远,但CVA4病毒株却保持着极近的亲缘关系。
基于CVA6VP1区全长核苷酸序列,我们将CVA6分为4个基因型,洛阳市3株CVA16均属于C基因型。他们之间保持着很高的核苷酸和氨基酸同源性。
由于可获得的CVA2VP1区核苷酸序列有限,利用Genbank可用的VP1区部分核苷酸序列,可将CVA2分为4个组,它们核苷酸同源性为76.9%-92.3%,但却有着97.8%以上的氨基酸同源性,表明它们在抗原性上保持着高度一致性。
对CVB2VP1区全长核苷酸序列的分析结果显示,可以将CVB2划分为3个基因型,分离自洛阳市的病毒株分布在C基因型,与2004年分离自山东的毒株保持着最高的核苷酸和氨基酸同源性,它虽然和A基因型的病毒株核苷酸同源性最低,但核苷酸却能保持很高的同源性,表明它们的抗原性并没有由于核苷酸的差异而发生大的改变。
基于CVB5的VP1区全长核苷酸序列,可将其分为6个基因组,分离自开封的病毒株属于D基因组,和我国分离到的CVB5处于同一个基因型,但是与别的病毒株核苷酸和氨基酸同源性均较低,显示其发生了较大的变异。
将ECH07分为4个基因型,同样是基于VP1区全长核苷酸序列。分离自开封的病毒株为C基因型,与分离自山东的病毒株亲缘关系最近。这也是第二次报道ECHO7可引起HFMD。
CVA24作为HEV-C组的成员,通常被认为是急性出血性结膜炎(Acute hemorrhagic conjunctivitis, AHC)的病原。通过对分离自开封HFMD患者标本2株CVA24和来自GenBank病毒株全长VP4区核苷酸序列的研究,不高的核苷酸序列和不高的氨基酸序列,显示该病毒的的VP4区基因相当活跃。虽然CVA24也被认为可以引起HFMD,但鲜有报道,将其作为HFMD的可能病原,还需要更多的工作去证实。
Hand, foot, and mouth disease (HFMD), a highly contagious enteroviral infection, usually affects infants and children.
Generally, HFMD is mild and self-limiting in children, the clinical symptoms showed vesicular exanthema on their hands, feet, mouths and buttocks. But severe cases usually presented with neurologic complications, brainstem encephalitis or aseptic meningitis; continuous high fever (temperature of at least38℃); weakness, vomiting, irritability, myoclonus and acute flaccid paralysis; pulmonary edema or hemorrhage, heart and lung failure. And Human enterovirus71(HEV71) and Coxsackievirus A16(CVA16) are the major causative agents of this disease.
In recent years, numerous large outbreaks of HFMD have been reported in Eastern and Southeastern Asian countries, including Singapore, Korea, Malaysia, Japan, Vietnam, and China. An outbreak of HFMD had occurred in Fuyang City of Anhui Province, China,2008. A total of6,049HFMD cases were reported between1March and9May in this outbreak, which included22fatal cases. Since the epidemic developed over a relatively short time span and was associated with high morbidity and mortality, HFMD had received considerable attention from clinicians and public health officials, and HFMD was classified as a category C notifiable infectious disease by the Ministry of Health of P. R. China on May2,2008. After20083, the outbreak of HFMD continues in China, and the severe or fatal cases were reported year by year.
In order to analyze the pathogens of HFMD, set up the database of their molecular character in Luoyang and Kaifeng city,327stool specimens were collected from327hospitalized children with characteristic symptoms of HFMD within7days of onset from March to June in Luoyang and Kaifeng city, the peak time of this outbreak.
The cell lines used to isolate viruses from the specimens were Human rhabdomyosarcoma (RD) cells and human laryngeal carcinoma (HEp-2) cells. If the second-generation cultures showed negative results, the nucleic acid hybridization was performed. The positive results of the hybridization led to the third-generation culture. The results showed that introduction of the nucleic acid hybridization had increased the rate of virus isolation. Finally, a total of230isolates from RD cell lines and8isolates from HEp-2cell lines were obtained.
The complete VP1or VP4region of the isolates was amplified by RT-PCR with specific primers, and serotype was identified by sequence analysis.
Among the isolates from Luoyang,118isolates were identified as HEV71, and26isolates were identified as CVA16. In addition, three isolates as CVA6, one isolate as Echovirus30(ECHO30), one isolate as CVA2, one isolate as CVA4, one isolate as CVA24and one isolate as CVB2were identified. And in Kaifeng's isolates,66isolates were identified as HEV71,10isolates as CVA16,2isolates as CVA24,2isolates as CVB5, one isolate as ECHO7and isolate as CVA4and three human adenoviruses (HAdV) were found in the isolates.
Exclude the interference of co-infection and non-HEV HFMD pathogens identified, the etiology of Luoyang City, Henan Province in2009included the HEV71(78.76%), CVA16(17.33%), CVA6(2.00%), CVA4(0.67%), CVA2(0.67%), CVB2(0.67%), in Kaifeng, HEV71(81.48%), CVA16(12.35%), CVA4(1.23%), CVB5(1.23%), ECHO7(1.23%), CVA24(1.23%).
Alignment of the nucleotide sequences of the isolates was performed, and a phylogenetic dendrogram was constructed. The complete VP1region sequences of the HEV71strains isolated from the patients were used for phylogenetic analysis. There were4transmission chains in Luoyang and2in Kaifeng at least. It was shown that all the HEV71strains isolated belonged to branch C4a of the subgenotype C4, similar to the HEV71sequences isolated from China during2003-2008.
The phylogenetic analysis of VP1region of CVA16showed that,2genotypes can be assigned, A and B. The genotype B could be divided into B1and B2, and subgenotype B1could be further divided into clusters B1a and B1b. All CVA16strains isolated in this study belonged to clusters B1a and B1b within the subgenotype B1. There were4transmission chains co-circulated in Luoyang and7in Kaifeng at least. It has high amino acid identity with the strains isolated from Luoyang and Kaifeng city.
The entire VP1sequences of CVA4were used for phylogenetic analysis. The results indicated that5genotypes could be divided, and the strains isolated from Luoyang and Kaifeng were in the same genotype:C. It indicated they maintained high homology of nucleotide and amino acid.
Based on complete VP1sequences of CVA6, it should be divided into4genotypes:A, B, C and D. The Luoyang strains were in the C genotype, and kept the high homology of nucleotide and amino acid.
Because of the absence of entire VP1sequences of CVA2in the Genbank database, the partial VP1sequences were used for constructing phylogenetic dendrogram. The results showed that they were divided into4clusters. The CVA2strain isolated from Luoyang was belonged to cluster2.
The phylogenetic analysis of VP1region of CVB2showed that,3genotypes could be assigned, Luoyang CVB2belonged to genotype C, and had high nucleotide similarity and amino acid similarity with strains isolated from Shandong province in2004. Although it had low nucleotide identity with genotype A viruses, it had high amino acid identity with them, which indicated that their antigenicity may not alter so much due to big differences in nucleotide.
6genotypes within CVB5could be assigned based on their complete VP1sequences, and Kaifeng CVB5belonged to genotype D, which clustered with other CVB5isolated in China. It had low nucleotide similarity and amino acid similarity with strains isolated in other countries, and showed big variation.
The phylogenetic analysis of complete VP1region of ECHO7showed that,4genotypes could be assigned. Kaifeng ECHO7belonged to genotype C, and had high nucleotide similarity and amino acid similarity with strains isolated from Shandong province in2008.
As a member of HEV-C, CVA24was reported to be a pathogen of acute hemorrhagic conjunctivitis (AHC) usually. But2isolates from the HFMD cases indicated it was probable agent of HFMD. In fact, CVA24was isolated in other outbreaks of HFMD, so it needs to be further confirmed.
引文
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