江西省儿童急性出血性结膜炎病原学监测研究

目的了解2011年-2012年江西省儿童急性出血性结膜炎病原学特征。方法采用PCR和逆转录PCR(RTPCR)检测方法,对2011年-2012年江西省急性出血性结膜炎患儿结膜拭子标本同时进行肠道病毒70型、柯萨奇病毒24型变异株和腺病毒3种病毒核酸检测。结果 2011年-2012年江西省儿童流行性出血性结膜炎各月份均有发病,均为年初开始发病,且呈逐月增多,于5月-7月最高。2011年结膜拭子标本病毒核酸阳性率为77.78%。其中,腺病毒占98.10%,肠道病毒70型占1.90%;2012年结膜拭子标本中病毒核酸阳性率为70.45%,均为腺病毒。结论 2011年-2012年江西省儿童急性出血性结膜炎流行高峰为5月-7月,病原谱构成为腺病毒占病毒阳性的比率上升,肠道病毒比率则下降。     

2010年湖南省郴州市手足口病病原学和人肠道病毒71型基因特征研究

目的:了解2010年湖南省郴州市手足口病的病原学特征,为手足口病的防控工作提供依据。方法:采集2010年郴州市手足口病患者的咽拭子、肛拭子或粪便标本,采用Real time-RT-PCR方法检测HEV71、CVA16和HEV的核酸,选择HEV71阳性的标本使用RD、Vero细胞进行病毒分离,使用RT-PCR法扩增HEV71分离株的VP1区片段,进行VP1区核苷酸序列测定和分析。结果:2010年共检测手足口病患者临床标本665份,HEV阳性率72.78%,其中HEV71占55.17%、CVA16占18.59%、其它HEV占26.24%。不同月份、年龄、地区以及不同类型病例HEV型别构成比差异有统计学意义。分离出4株HEV71,其VP1区核苷酸序列分析表明为C4基因亚型。结论:HEV71为郴州市2010年手足口病流行的主要病原体,属于C4基因亚型。HEV71和CVA16的分布在病例发病时间,发病年龄和地域上有所不同。     

合肥市某小学急性呼吸道感染疫情的病原学分析

目的分析合肥市某小学一起急性呼吸道感染疫情病原学,为疫情发生的生物学病因提供依据。方法采用实时荧光PCR方法(Real-time PCR)对采集的10份患者咽拭子标本进行流感病毒和腺病毒核酸筛查,检出的腺病毒核酸阳性标本经普通PCR扩增腺病毒六邻体基因片段以进行序列测定,测序结果在Genbank上进行Blast比较并作系统进化树分析,以确定其病毒型别。结果 10份咽拭子标本流感病毒核酸检测结果均为阴性,7份标本检出腺病毒核酸,进一步通过基因测序、序列比对、基因进化树分析确定为腺病毒3型。结论依据实验室检测结果并结合流行病学调查和临床症状,确认该起急性呼吸道感染疫情的病原体为人腺病毒3型。     

人腺病毒7型引起的呼吸道感染疫情病原分子生物学分析

目的对安庆市某部队一起人腺病毒7型引起的急性呼吸道感染暴发疫情进行病原学鉴定,并对其六邻体(Hexon)基因进行测序分析。方法采取Real-time PCR对采集的13份咽拭子标本进行腺病毒核酸检测,阳性标本接种Hep-2细胞进行病毒分离培养。收集阳性分离培养物,采用RT-PCR扩增Hexon的核苷酸序列进行测序,获得序列在NCBI GenBank数据库进行同源比对,MAGE6.0软件进行进化分析、构建进化树。结果获得10份阳性毒株培养物,Hexon基因序列与我国武汉7型腺病毒疫情株Z9/WH/CHN/2015(GenBank号为KX897164)100%同源,与2012AQHAdv7疫情株和2015AQHAdv7散发株100%同源性。结论该起疫情由人腺病毒7型感染引起,安庆地区流行的腺病毒疫情株基因型别稳定,此次的疫情可能源于本地的流行株。     

一起由腺病毒和肠道病毒混合感染引起的发热呼吸道感染暴发疫情的实验室快速诊断

目的对一起婴幼儿不明原因发热呼吸道感染暴发疫情进行实验室快速诊断,探索病因。方法采集患儿咽拭子和痰液标本共19份,对所有标本进行核酸提取、运用荧光PCR和荧光RT-PCR法检测腺病毒等引起急性呼吸道感染的8种18型重要病毒的特异性基因片段。对腺病毒和肠道病毒核酸阳性标本进行序列测定,运用生物学软件对测定序列进行处理分析。结果 19份标本腺病毒(Adenovirus,Ad V)核酸阳性,11份标本肠道病毒(Enterovirus,EV)核酸阳性,4份标本鼻病毒(Rhinovirus,RHV)核酸阳性。Ad V的核酸检出阳性率均为100%,标本采集时间最长为发病后10天。EV在发病3天内痰液标本中核酸检出率为83.33%,发病3天后为42.86%。Ad V的基因型别为Ad V-B7型。结论本起发热呼吸道感染暴发疫情由Ad V-B7型和EV混合感染引起,并且Ad V-B7型感染时间长于EV。     

一起人腺病毒14型所致成年人不明原因肺炎的病原学检测分析

摘要:目的　分析一起导致一名成年人不明原因肺炎的病毒性病原体,确立病原微生物载体与病人之间的相关性,为临床防治提供实验室依据。方法　将采集到的呼吸道标本采用实时荧光定量PCR（Real-time PCR）检测流感病毒、冠状病毒、副流感病毒、呼吸道合胞病毒、人偏肺病毒、人博卡病毒、人腺病毒和鼻病毒核酸,对检测结果为腺病毒阳性的核酸进一步采用腺病毒特异性引物通过PCR进行Hexon,Fiber和E1A区基因特异性扩增,产物进行核苷酸序列测定和比对分析并构建遗传进化树。结果　1份咽拭子和1份支气管灌洗液核酸经Real-time PCR检测均呈腺病毒阳性,扩增病毒基因序列经测序分析与人腺病毒14型同源性最高。结论　此起成年人不明原因肺炎是由人腺病毒14型感染所致。     

2011 - 2017年济南市呼吸道腺病毒流行特征及其基因分型

目的 监测2011 - 2017年山东济南急性呼吸道感染(acute low respiratory tract infection,ALRTI)中腺病毒（Human Adenovirus, HAdV）的感染情况,揭示济南地区不同年份、不同季节腺病毒感染的流行病学特征和基因进化树特点。方法 2011 - 2017年在济南市中心医院、山东大学齐鲁儿童医院共采集1 730份急性呼吸道感染住院病例鼻、咽拭子标本,采用荧光定量PCR方法对标本进行腺病毒检测。检测为腺病毒阳性的标本再进行病毒分离、PCR扩增、Hexon测序、Blast比对,构建基因进化树,确定腺病毒型别,分析其进化特点。结果 在收集的1 730份标本中,共102份确定为腺病毒核酸阳性,阳性率为5.90%,其中2011年阳性率最高,为14.78%,2012年阳性率最低,为2.34%;阳性率最高的为1月份,达到10.33%,最低的为8月份,为1.91%;阳性率最高的为5～15岁年龄组,为9.76%,最低的为≥60岁年龄组,为1.37%。从102份腺病毒核酸阳性标本中分离出50株腺病毒,分子分型共检出5个血清型,其中最多的为3型,占54%,其次为7型、55型、2型和5型,分别占32%、8%、4%和2%,基因进化分析没有发现相关变异。结论 2011 - 2017年山东济南地区腺病毒感染以3型为主,其次是7型,而55型、2型和5型较少检出。     

一起腺病毒3型引起暴发疫情的调查

目的对发生在富阳市渔山乡小学的一起上呼吸道感染暴发疫情进行流行病学调查和病原学鉴定。方法对该暴发疫情进行流行病学调查和采集现症病例咽拭子。应用实时RT-PCR和实时PCR技术分别进行流感病毒和人腺病毒检测。对人腺病毒核酸阳性标本,应用PCR技术扩增六邻体基因超变区7的核酸片段并进行核苷酸序列测定,根据序列相似性以鉴定血清型。结果 2013年5月20—28日共有21例上呼吸道感染病例,均集中在二（1）班,全校罹患率为4.77%,班级罹患率高达70.00%。病例均有发热,部分有咳嗽、咽痛、腹痛、腹泻和结膜充血等症状。检测的8份咽拭子中,实时PCR检测腺病毒核酸阳性5份,甲型流感病毒（通用型）、H1N1、H3N2、H7N9及乙型流感病毒核酸的实时RT-PCR检测结果均为阴性。选取其中1份阳性标本,进行人腺病毒六邻体基因超变区7的核酸序列测定,结果与近年国内其他地区分离的多株腺病毒3型毒株的相似性为100.00%,因此鉴定为腺病毒血清型3型。结论结合流行病学调查、病例临床症状及实验室检测结果,判断该起呼吸道感染暴发疫情的病原体为人腺病毒血清型3型。     

一起由3型腺病毒引起的咽结膜热病原学研究

目的　探讨2 0 0 4年7月上旬广州市某幼儿园暴发的一起咽结膜热的病原学。方法　采集急性咽结膜热患儿急性期眼或咽拭子标本用Hep 2细胞进行病毒分离,并对原始标本及病毒分离物使用PCR方法及序列测定进行检测鉴定。用分离鉴定的毒株作为抗原,测定7例患儿及2名健康儿童(阴性对照)双份血清中病毒的中和抗体。结果　2 2份患儿的咽拭子和眼拭子标本用PCR测定显示12份为腺病毒阳性,2 2份标本中只分离出9株毒株,经鉴定所有病毒株均为3型腺病毒,且与朝鲜分离的3型腺病毒同源性高达97%。以分离到的1株毒株与7例患儿自身急性期和恢复期血清的中和试验显示恢复期中和抗体较急性期有4倍或4倍以上增长,2例阴性对照中和抗体无增长。结论　该起急性咽结膜热暴发是由3型腺病毒引起的     

2009—2011年上海市宝山区手足口病病原学研究

[目的]探讨2009—2011年上海市宝山区手足口病病原学特征及诊断意义。[方法]2009年3月—2011年12月连续采集手足口病临床诊断病例的咽拭子、大便样本或肛拭子和疱疹液进行肠道病毒71型(enterovirus 71,EV71)、柯萨奇病毒A组16型(coxsackievirus A16,CoxA16)和其他肠道病毒核酸检测。[结果]2009—2011年肠道病毒的总检出率分别为59.00%、72.44%和79.00%,重症(死亡)病例中EV71型肠道病毒的阳性检出率为90.91%,明显高于普通病例(χ2=45.97,P<0.001)和聚集性病例的检出率(χ2=56.85,P<0.001);咽拭子、肛拭子或大便样本、疱疹液、咽漱液标本肠道病毒阳性检出率分别为68.02%、86.67%、71.43%、25.00%;咽拭子标本与肛拭子或大便标本和疱疹液标本检测结果符合率为83.53%和100.00%;发病0~4d肠道病毒核酸检出率为79.73%,5~10d肠道病毒核酸检出率为45.61%,差异有统计学意义(χ2=30.91,P<0.001)。[结论]2009—2011年宝山区手足口病感染的主要病原是EV71型和CoxA16型肠道病毒,肠道病毒的检出率逐年提升,且每年的优势型别有所不同;手足口病重症(死亡)病例的主要病原为EV71型肠道病毒;咽拭子、肛拭子或大便样本、疱疹液都有较高的诊断价值;病后4d内采集标本对手足口病诊断效果较好。     

Molecular Classification of Human Adenovirus Type 7 Isolated From Acute Respiratory Disease Outbreak (ARD) in Korea, 2005–2006

Objectives

To assess the genomic characteristics of human adenoviruses (HAdVs) that caused small-scale epidemics in Korea and compare sequence analysis and restriction fragment length polymorphism (RFLP).

Methods

Two hundred sixty-two throat swabs were collected from geographically distinct two cohabitation facilities during outbreaks in August 2005 and February–May 2006. 148 isolates were obtained using the adenocarcinomic human alveolar basal epithelial cells (A549 cells) from 262 specimens. The sequences of 448 bp partial hexon gene of isolates were analized and compared with serotype results using neutralizing test. The hexon (1.2 kb), fiber, and E4 ORF 6/7 34.7 kDa protein (2.1 kb) genes were further analysed in 10 randomly selected specimens. RFLP of the genomic DNA for genotyping was also performed and compared with sequence information.

Results

All the isolates were localized into the same cluster when phylogenetic tree was generated based on hexon gene using Clustal W. While fiber and E4 ORF 6/7 34.7 kDa protein genes were analysed, the tree was divided into two clusters. Interestingly, isolates with same genetic characteristics of hexon gene did not show identical RFLP patterns in accordance with their origin of episode, rather phylogenetic analysis of fiber and E4 ORF 6/7 34.7 kDa protein genes were correlated with RFLP patterns.

Conclusion

These results indicate that serotype classification based on hexon gene might not be enough to discriminate HAdV serotype, and additional genetic characteristics including fiber and/or E4 ORF 6/7 should be recruited to dispose subgroup of HAdV serotype.Key words: E4 ORF 6/7, fiber, hexon, human adenovirus Type 7, restriction fragment  length polymorphism     

Molecular detection of adenovirus type-8 epidemic keratoconjunctivitis in Hungary

INTRODUCTION: Both infectious and non-infectious forms of acute conjunctivitis are known. Viruses, especially different types of adenoviruses are the etiological agents of infectious epidemic conjunctivitis (conjunctivitis epidemica). AIMS: The author's aims were to describe an outbreak of keratoconjunctivitis and to detect the viral agent by molecular methods in Hungary. MATERIALS AND METHODS: Classical epidemiological methods were used for investigation. Polymerase chain reaction (PCR) followed by sequencing were used for the detection of adenoviral hexon region from freshly collected conjunctival swabs. RESULTS: Between 9 October and 18 December 2006, a total of 60 patients became ill with keratoconjunctivitis in 7 settlements in Southwest Hungary. Mean age was 51,2 years. Conjunctivitis (100%), lacrimation (94%), foreign body sensation (83%), and dim vision (76%) were the main clinical symptoms. Both eyes were affected in half of the cases. Direct contact was the main transmission route including nosocomial spread associated with ophtalmological practices. Five (62.5%) of 8 conjunctival swabs were PCR-positive for adenovirus type 8 (HAdV8/Baranya/2006/HUN; EF210714) which was genetically identical to adenovirus strain detected in Austria in 2004 (DQ149614). CONCLUSIONS: The outbreak of keratoconjunctivitis was partially associated with nosocomial infection caused by type 8 adenovirus. Both the recognition of the clinical illness, laboratory diagnosis and public health measures are necessary for the prevention of keratoconjunctivitis infection and epidemic.     

Detection of airborne viruses in a pediatrics department measured using real-time qPCR coupled to an air-sampling filter method

Children are vulnerable to viral infections. The study discussed in this article investigates the possibility of aerosol transmission of viruses in children under age 18 in the pediatrics department of a medical center in Taipei, Taiwan. After first using the filtration method to collect viral aerosols, the authors combined it with real-time quantitative polymerase chain reaction (qPCR) to detect influenza A virus (INFAV), human adenovirus (HAdV), and enterovirus. Of 33 aerosol samples collected from the emergency room of the pediatrics department, 8 (24%) were positive for INFAV, 12 (36%) for HAdV, and 5 (15%) for enterovirus. HAdV was detected from the aerosol of 26 samples, but INFAV and enteroviruses were not. The filter and real-time qPCR can be used to detect and quantify the viral load in aerosols, in which enteroviruses had the highest viral titer. In summary, a well-established filter/real-time qPCR assay was successfully applied to measure viral aerosols in the occupational environment. Environmental monitoring of airborne viruses could provide an early indicator of dangerous viruses in the air of hospitals.     

贵州省从江县81例手足口病病例病原学检测结果

目的了解贵州省从江县2011—2012年手足口病流行的主要病原体,为手足口病防控提供科学依据。方法采集临床诊断为手足口病病例的咽拭子采用RT-PCR方法扩增病毒的特异性片段,检测EV71、CoxA16核酸和其他肠道病毒核酸。结果共采集标本81份,病毒核酸检测阳性33份,阳性率为40.74%;其中EV71阳性9份（27.27%）,CoxA16阳性4份（12.12%）,其他肠道病毒阳性20份（60.61%）。结论从江县2011—2012年手足口病流行毒株以其他肠道病毒为主。但也不能忽视EV71引起的有中枢神经系统并发症的重症病例的监测。     

手足口病EV71和CoxA16检出率的影响因素分析

目的了解厦门市手足口病病原检出率的影响因素。方法 2009年至2010年,收集哨点医院病例的咽拭子和血清标本1 502人份,用荧光定量RT-PCR法进行核酸检测,分析EV71和CoxA16检出率的影响因素。结果 EV71和CoxA16总检出率为63.5%（954/1 502）。咽拭子检出率高于血清;发病3 d内的标本检出率较高,时间间隔越久检出率越低;专科医院检出率高于综合医院;年龄别检出率呈抛物线形。结论标本类型、采集时间、医院类型、患者年龄与EV71和CoxA16检出率相关。     

An outbreak of acute haemorrhagic conjunctivitis caused by enterovirus 70 in Jeddah during 1985

Seventy-five cases of acute haemorrhagic conjunctivitis (A.H.C.) were subjected to full ophthalmic, bacteriological, virological, serological as well as cytological examinations. The majority of cases presented with bilateral conjunctivitis (70 out of 75). Although follicular reaction was the earliest sign, yet subconjunctival haemorrhages were the most constant findings in all the cases. Enterovirus 70 was isolated in 57 cases from conjunctival swabs and in 43 cases from serum samples. Complement flaxation test was positive for enterovirus 70 in 42 cases but negative for coxsackie and adenoviruses. No primary role has been found for bacteria in the pathogenesis of A.H.C. in this outbreak. Cytological examination of conjunctival scrappings showed the characteristics cytopathic effect (CPE) of enteroviruses.     

杭州市急性出血性结膜炎的柯萨奇病毒分离鉴定及其VP1序列分析

目的:2010年杭州市发生了两起急性出血性结膜炎(Acute Hemorrhagic Conjunctivitis,AHC)疫情,为鉴定引起疫情的致病病原体。方法:采集了9份眼结膜拭子标本,使用实时荧光PCR或RT-PCR法分别检测临床标本中腺病毒、人肠道病毒70型(Human Enterovirus 70,HEV70)和柯萨奇病毒A组24型变种(CoxsackievirusA24 variant,CVA24v)的基因。使用Hep-2细胞和RD细胞分离病毒。对分离到的CVA24v毒株进行全长VP1区扩增和核苷酸序列测定和分析。结果:实时荧光PCR或RT-PCR法检测临床标本,其中有6份检测结果为CVA24v阳性,阳性率为66.7%,而腺病毒和HEV70检测结果均为阴性。使用Hep-2细胞共分离到5株病毒,通过分子定型均鉴定为CVA24v。进化树图显示,两起疫情是由两个病毒传播链引起的。结论:本研究成功地分离到5株CVA24v毒株,为今后杭州市AHC的防治及研究奠定了一定的基础。     

Rapid identification and metagenomics analysis of the adenovirus type 55 outbreak in Hubei using real-time and high-throughput sequencing platforms

The rise in human adenovirus (HAdV) infections poses a serious challenge to public health in China. Real-time (RT) sequencing provides solutions for achieving rapid pathogen identification during outbreaks, whereas high-throughput sequencing yields higher sequence accuracy. In the present study, we report the outcomes of applying nanopore and BGI platforms in the identification and genomic analysis of an HAdV outbreak in Hubei province, China in May of 2019. A mixed sample of nine nasopharyngeal swabs and one single sample were submitted to direct nanopore sequencing (MinION device), generating their first HAdV-55 reads within 13 and 20 min, respectively. The sequences were confirmed by RT-polymerase chain reaction (PCR). Ten HAdV-positive samples were further sequenced using next-generation high-throughput sequencing (BGISEQ-500 device). Phylogenetic analysis revealed that the outbreak strain had a close genetic relation to strains isolated in Sichuan province. Metagenomic analysis showed that HAdV-55 was not a dominant species in samples from which the whole HAdV-55 genome could not be assembled. The present results highlight the value of combining sequencing platforms and using mixed samples for nucleic acid enrichment in pathogen detection of infectious disease outbreaks.     

Isolation of enterovirus 70 (EV70) from patients with acute haemorrhagic conjunctivitis in two areas of Saudi Arabia

This is the first epidemic of acute haemorrhagic conjunctivitis (AHC) reported from Saudi Arabia in which enterovirus 70 (EV70) was implicated as aetiological agent. EV70 was isolated from 5 of 29 conjunctival scrapings from patients with AHC. Two human diploid cell lines, human skin fibroblast (HSF) and human embryonic lung fibroblast (MRC-5), were quite sensitive for the isolation of this virus. The relatively high isolation rate could also be attributed to the timing of collection of specimens and perhaps to the fact that conjunctival scrapings contained more virus particles than did eye swabs.