南京地区儿童人偏肺病毒感染的流行病学及临床特征分析

目的了解南京地区儿童人偏肺病毒（hMPV）感染的流行病学特点及临床特征。方法收集2009年8月至2010年7月南京医科大学附属南京儿童医院住院及门诊呼吸道感染患儿的鼻咽抽吸物（NPA）及咽拭子（NPS）共642例,采用逆转录聚合酶链反应法（RT—PCR）检测hMPVM基因,将阳性PCR扩增产物进行测序、同源性和进化分析。结果642例标本中共检出hMPV阳性扩增产物35份,检出率为5．5％。系统进化分析显示南京地区hMPVB1型占51．4％,A2b型占31．4％。hMPV的发病高峰在4月份。其致呼吸道感染以1岁以内多见（71．4％）。35例hMPV感染患儿中有15例（42．8％）存在混合感染,其中与HRV的混合感染检出率最高。临床诊断以肺炎（17例,48．6％）最为常见。结论人偏肺病毒是南京地区儿童急性呼吸道感染的重要病原之一,该年度其优势流行型别为B1型,南京地区A、B两型hMPV感染患儿临床特征无明显差异。     

上海地区儿童下呼吸道人类偏肺病毒感染的初步研究

目的 了解人类偏肺病毒（hMPV）在上海地区儿童下呼吸道感染中的致病情况。方法 采集2004年8月-2005年1月秋冬季节我院479例因社区获得性急性下呼吸道感染（CALRTIs）住院儿童的呼吸道分泌物标本，对直接免疫荧光法常规检测病毒阴性的259份标本用逆转录．多聚酶链反应法（RT-PCR）检测hMPVM基因。对PCR阳性产物随机挑选23份直接作核苷酸序列测定，用DNAStar软件对基因序列分析。结果 259份标本中hMPV检测阳性例数为59例（22．8％），占总例数的12．3％。冬季检测阳性率明显高于秋季（31．3％vs7．5％，P〈0．01）。5岁及以下儿童53例（89．8％），5岁以上儿童6例（10．2％）。23份hMPV阳性标本目标基因部分核苷酸序列与GenBank中公布的hMPVM基因同源性达82．8％～100％，部分氨基酸序列同源性为93．0％～100％，对核苷酸序列作基因进化树分析显示存在2种不同的基因型，以Bj1816组为代表的基因型14株，以Bj1887组为代表的基因型9株。结论hMPV是上海地区儿童CALRTIs的重要致病原，上海地区儿童感染的hMPV同时存在2种不同的基因型。     

小鼠人偏肺病毒感染模型的建立

目的 建立人偏肺病毒(hMPV)感染小鼠模型,了解病毒肺内复制规律及所致病理改变,为hMPV感染免疫病理机制研究及新型防治手段开发奠定基础.方法 BALB/c小鼠经滴鼻感染荧光标记的重组hMPV,于感染后1、3、5、7、9、16 d处死小鼠并无菌获取肺组织用于病毒分离和病理检查,改良噬斑形成法检测hMPV滴度,RT-PCR法检测hMPV mRNA表达.结果 小鼠滴鼻感染hMPV后肺组织分离到病毒;肺组织病毒滴度在感染后5 d达到高峰(5.16±1.09)×105PFU/g,感染后第9大仍能检测到病毒(2.79±1.22)×102PFU/g;感染后16 d肺组织仍可检测到hMPV mRNA;病理改变在感染后3～7 d最明显,为典型的间质性肺炎改变.结论 hMPV感染BALB/c小鼠模型建立成功,可用于hMPV感染的免疫病理机制研究.     

2007—2011年长沙地区急性下呼吸道感染住院儿童人偏肺病毒的流行状况

目的了解人偏肺病毒（hMPV）在长沙地区急性下呼吸道感染住院患儿中的流行病学特点。方法收集2007年9月至2011年2月因急性下呼吸道感染在湖南省人民医院儿科医学中心住院儿童的鼻咽抽吸物（nasopharyngealaspirates,NPA）样本2613份,采用逆转录聚合酶链反应法（RT—PCR）检测hMPVM基因,将阳性PCR扩增产物测序并与GenBank中已知的hMPV参考株进行比对、分析。结果2613份标本中hMPV阳性检出数135例,检出率为5．2％,男女之间的检出率比较有统计学差异（x2=8．007,P=0．003）,hMPV阳性检出患儿的年龄以1岁以内多见（63．2％）。hMPV阳性检出率在春季呈现高峰,从检出季节分布显示A2b型主要在冬春季节流行,而B2型主要在春夏季流行。135例hMPV长沙株分为A型和B型两个主要的基因型,其中A2b亚型在2007--2008年为优势流行型别,2009--2010年A2b和B2型共同流行,B2亚型在2011年呈优势流行型别。135例hMPV检出阳性患儿中有66例（48．9％）存在混合病毒感染,其中与HBoV混合检出率最高。结论长沙地区部分儿童的急性下呼吸道感染与hMPV有关,且阳性检出患儿年龄主要集中在1岁以下,男多于女,主要流行季节在春季,A2b型和B2型优势基因型在长沙地区共同流行,与其他病毒混合检出率较高。     

北京地区人偏肺病毒核蛋白基因的原核表达及抗原活性分析

目的用大肠杆菌表达获得人偏肺病毒主要结构蛋白N蛋白，为下一步的深入研究奠定基础。方法从重组质粒pUCm-N1816中PCR扩增获得N基因，用BamHⅠ和EcoRⅠ双酶切后插入到原核表达载体pET30a（＋）中，得到重组表达质粒pE130a-N1816，转化大肠杆菌BL21（DE3），IPIG诱导培养。SDS-PAGE和Western blot检测目的蛋白的表达和抗原性。结果经双酶切和测序证明1．2kb N1816。基因正确插入pET30a中，并具有正确的读码框架，得到预期的pET30a-N1816重组原核表达质粒。37℃，1mmol／L IFIG诱导培养6h后产生大量带6个组氨酸标记的重组N蛋白，主要以包涵体形式存在，占细胞总蛋白的20％左右。N蛋白粗提物经Co^2＋亲和层析获得较理想纯化。Western blot结果显示，体外所表达的蛋白能和特异性抗血清及人血清反应。结论本研究成功构建了重组pET30a-N1816原核表达质粒，N蛋白获得了高效表达，并且具有特异抗原活性，可用于人偏肺病毒的深入研究。     

玉屏风散对小鼠哮喘合并人偏肺病毒感染的干预机制探讨

目的:建立人偏肺病毒(Human metapneumovirus,hMPV)感染的哮喘小鼠模型,验证玉屏风散对hMPV诱导的哮喘小鼠气道高反应性和炎症的疗效并初步探讨其机理。方法:随机将48只雌性BALB/c小鼠分为对照组(A组)、OVA致敏激发哮喘组(B组)、偏肺病毒哮喘组(C组)、小剂量玉屏风组(D组)、中剂量玉屏风组(E组)、大剂量玉屏风组(F组),采用卵蛋白致敏和激发,hMPV滴鼻方法建立哮喘人偏肺病毒感染模型并给予玉屏风治疗,采用动物体描箱法测定气道反应性;采用支气管肺泡灌洗液(BALF)进行细胞分类计数;肺组织病理切片HE染色观察炎性细胞浸润;采用流式细胞术分别测定肺部细胞因子IFNγ-和IL-4、IL-17。结果:(1)随吸入乙酰甲胆碱浓度增加各组气道反应性明显增加,C组比B组气道反应性显著升高(P<0.05);F组气道反应性明显降低,低于C组(P<0.05);(2)各组BALF中白细胞总数及炎症细胞数都比对照组显著升高,C组BALF中白细胞总数、嗜酸性粒细胞与B组、F组相比显著升高(P<0.05);(3)各哮喘组的炎症评分都显著高于对照组。在细支气管周围炎、肺泡炎方面B组和F组比C组明显减轻(P<0.05)。(4)A、B组IFNγ-无差异(P>0.05),各剂量组IFNγ-,IFNγ-/IL-4均显著高于B、C组(P<0.05);哮喘组、治疗组IL-4显著高于正常组(P<0.01),但各组间无明显差异;与B组相比,C组IL-17水平明显升高(P<0.05);与C组相比,E、F组IL-17水平明显降低。结论:大剂量玉屏风散可以抑制感染hMPV哮喘小鼠的气道高反应性及气道炎症,且改善Th1/Th2失衡状态。     

中国湖南地区人偏肺病毒的基因分型及其膜蛋白G基因特征的分析

目的 研究中国湖南地区人偏肺病毒(hMPV)的基因分型及其膜蛋白G序列的遗传学特征,比较分析其区域性流行特点.方法收集2005年中国湖南地区232份住院儿童鼻咽抽吸物(NPA)样本进行常见呼吸道病毒筛查,并扩增hMPV阳性样本的膜蛋白G全序列,与GenBank中已知hMPV参考株及其他地区公布的相关资料进行比对、进化等分子遗传学特征分析.结果从232份临床样本中共检测得到17份(7.3%)hMPV阳性样本,与其他病毒混合感染率达35%.扩增出其中13份hMPV样本的G蛋白全序列,分属于4种亚型(A1、A2、B1、B2).核酸长度类型有4种(711,675,660,696nt),N-连接糖基化位点数目和位置、半胱氨酸残基数目等特征与已报道的同期北京地区、日本、北美等地区hMPV调查分析结果不尽一致.结论中国湖南地区与其他地区同期hMPV调查分析结果各有特点,反映出hMPV变异显著,具有明显的地区流行特征.     

长沙地区住院儿童人冠状病毒NL63的感染状况及临床特征

目的 了解HCOV-NL63在长沙地区急性下呼吸道感染住院儿童中的流行状况以及临床特征.方法 采集长沙地区2008年9月至2010年10月因急性下呼吸道感染住院儿童NPA样本1185份,用RT-PCR方法检测HCOV-NL63N基因,阳性扩增产物经测序确认,统计分析临床资料.结果 1185份NPA样本中,HCOV-NL63检出阳性10份,检出率为0.8％,其中春季检出1例、夏季检出6例、秋季检出2例、冬季检出1例;阳性检出病例患儿年龄最大2岁半,最小2个月,平均月龄12.5个月;男童7例,女童3例;临床诊断为支气管肺炎6例、毛细支气管炎3例、急性喉气管支气管炎1例;10例中有4例病情危重;4例有基础疾病;7例合并其他病毒混合感染;阳性扩增产物经测序分析与HCOV-NL63参考株同源性为97％～100％.结论 本资料HCOV-NL63阳性检出患儿年龄均在3岁以下;夏季检出率高;男童感染为主;单一基因型在长沙地区流行;HCOV-NL63可能也是我国儿童急性下呼吸道感染中的一种病原.     

北京地区人偏肺病毒融合蛋白F编码基因的序列分析

目的进一步了解北京地区人偏肺病毒（hMPV）编码基因的特征。方法在原先工作的基础上，从分属于hMPV的两个不同基因进化簇（即基因型）的两份临床标本BJ1816和BJ1887中扩增F蛋白全基因，克隆至pBS-T载体中并进行测序，与GenBank中hMPV的基因序列进行比较分析和种系进化分析。结果BJ1816和BJ1887的F基因全长均为1620个核苷酸，编码539个氨基酸。BJ1816和BJ1887F蛋白基因之间的核苷酸和推导的氨基酸同源性分别为83．8％和94．4％，与同一基因簇内的hMPV有相当高的氨基酸同源性（98．3％～99．6％）。BJ1816和BJ1887的F蛋白是典型的I型糖蛋白，具有与迄今已发现的hMPV相同的裂解位点（RQSR）。BJ1816和BJ1887之间F2亚单位的氨基酸同源性高于F1亚单位的同源性（96．9％vs93．8％）；除位于羧基末端的跨膜区和胞质尾区外，F1亚单位的其余部分较保守（95．4％）；糖基化位点保守。种系进化分析显示BJ1816和BJ1887属于不同的进化簇。结论F蛋白的序列分析进一步证明BJ1816和BJ1887分属于不同的基因型，其F蛋白的基因特征与其他国外文献中所报道的hMPV相似，是病毒的膜表面糖蛋白，提示其在病毒的感染与免疫中起到重要的作用。     

北京新发现的人偏肺病毒N蛋白编码基因的序列分析

目的　了解新近从北京地区发现的人类偏肺病毒 (humanmetapneumovirus ,HMPV)N蛋白编码基因的特征。方法　从经RT PCR检测HMPV阳性的临床鼻咽洗液标本中进行HMPVN蛋白全基因扩增、克隆至pUCm T载体中并进行测序 ,与GenBank中的多株HMPVN蛋白基因序列进行比较和进化树分析。结果　经扩增并测序 ,标本BJ1816和BJ1887N蛋白基因全长为 1185bp ,编码 394个氨基酸。BJ1816N蛋白基因与国际上第一株HMPV分离株NDL0 0 1和GenBank中其它的多株HMPV的核苷酸和氨基酸同源性分别为 86 .2 %～ 99.0 %和 96 .2 %～ 99.7%。BJ1887N蛋白基因与以上进行比较的各株HMPV的核苷酸和氨基酸同源性分别为 86 .6 %～ 97.0 %和 96 .4 %～ 99.5 %。BJ1816和BJ1887之间N基因的核苷酸和氨基酸同源性分别为 86 .6 %和 96 .4 %。提示BJ1816和BJ1887分属于HMPV的两个不同的基因进化簇。结论　从基因水平进一步证明新近发现的婴幼儿肺炎的新病原确为HMPV ,提示北京地区婴幼儿中同时存在两个不同进化簇 (即基因型 )的HMPV感染。     

Paediatric human metapneumovirus infection: Epidemiology,prevention and therapy

Since its discovery in 2001, human metapneumovirus (hMPV) has been identified as one of the most frequent causes of upper and lower respiratory tract infections. Although a considerable number of hMPV infections are diagnosed in adults and the elderly, the highest incidence of infection is among children as seropositivity for hMPV approaches 100% by 5–10 years of age. Most of the diseases due to hMPV are mild or moderate, tend to resolve spontaneously, and only require outpatient treatment. However, some may be severe enough to require hospitalisation or, albeit rarely, admission to a paediatric intensive care unit because of acute respiratory failure. Mortality is exceptional, but may occur. The most severe diseases generally affect younger patients, prematurely born children, and children who acquire nosocomial hMPV infection and those with a severe chronic underlying disease. Global hMPV infection has a major impact on national health systems, which is why various attempts have recently been made to introduce effective preventive and therapeutic measures; however, although some are already in the phase of development (including vaccines and monoclonal antibodies), there is currently no substantial possibility of prevention and, despite its limitations, ribavirin is still the only possible treatment. Given the risk of severe disease in various groups of high-risk children and the frequency of infection in the otherwise healthy paediatric population, there is an urgent need for further research aimed at developing effective preventive and therapeutic measures against hMPV.     

Human metapneumovirus (hMPV) infection in immunocompromised children

BackgroundHuman metapneumovirus (hMPV) is a major cause of upper and lower respiratory tract infection (URTI, LRTI) in children. The prognosis of hMPV is unclear in immunocompromised patients.ObjectivesTo describe the characteristics of hMPV infection in immunocompromised pediatric patients and to review the literature.Study designThis retrospective study included 39 immunocompromised children (age 0–18 years) with proven hMPV infection attending two tertiary pediatric medical centers in 2004–2014. Demographic, clinical, laboratory, and radiological data were collected from the medical files.ResultsMedian age was 6 years. Seven patients had primary immune deficiency and 32, secondary immune deficiency, including 9 patients who underwent hematopoietic stem cell transplantation (HSCT). Most cases (92%) occurred in January–May. Twenty patients (51%) had lower respiratory tract infection and 17 (44%), upper respiratory tract infection; 2 patients (5%) had fever only. Presenting symptoms were fever (70%), cough (54%), and rhinorrhea (35%). Severe lymphopenia (<1000 lymphocytes/mL) was noted in 64% of patients and elevated liver enzyme levels in 49%. Seventeen patients had pneumonia: bilateral and alveolar in 13 patients, each. HSCT was not associated with more severe disease. Respiratory failure occurred in 6 patients, of whom 4 died (10% of cohort). All children who died had severe lymphopenia. On multivariate analysis, bacterial or fungal co-infection was the only major risk factor for death. Review of the literature showed variable clinical presentations and severity in pediatric patients with hMPV infection.ConclusionsInfection with hMPV may be associated with relatively high morbidity and mortality in immunocompromised children. Death was associated with bacterial and fungal co-infection.     

Prevalence of human metapneumovirus among hospitalized children younger than 1 year in Catalonia, Spain

Human metapneumovirus was discovered recently respiratory virus implicated in both upper and lower respiratory tract infection. In children, the clinical symptoms of human metapneumovirus are similar to those produced by respiratory syncytial virus, ranging from mild to severe diseases such as bronchiolitis and pneumonia. The aim of the present study was to describe the prevalence of human metapneumovirus and other common respiratory viruses among admitted to hospital infants. From January 2006 to June 2006, 99 nasopharyngeal aspirates were collected from hospitalized children younger than 12 months in order to study respiratory viruses. Human metapneumovirus detection was performed by cell culture and two RT-PCR targeting on polymerase and fusion genes. The latter gene was used for phylogenetic analysis. In 67/99 children (67%) at least one viral pathogen was identified, the viruses detected most frequently were respiratory syncytial virus (35%), human metapneumovirus (25%) and rhinovirus (19%). The results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among children less than 12 months; (2) children infected with human metapneumovirus were significantly older than those infected by respiratory syncytial virus; (3) human metapneumovirus was associated more frequently with pneumonia whereas respiratory syncytial virus was only detected in patients with bronchiolitis; (4) there was a clear epidemiological succession pattern with only a small overlap among the viruses detected most frequently; (5) all human metapneumovirus samples were clustered within sublineage A2.